Tuberous sclerosis, angiomyolipoma and lymphangioleiomyomatosis (LAM) are diseases characterised by slow growing tumours that are affecting many parts of the body (1, 2) including the skin, brain, kidneys and the lungs. The above diseases are caused by the mutation of tumour suppressor genes tuberous sclerosis 1 or 2 (TSC or TSC2) (1). The above tumours were initially considered benign, but angiomyolipoma and LAM have recently been reclassified as “low grade, destructive, metastasizing neoplasms” characterised by α-smooth muscle actin (α-SMA), vimentin, desmin and melanoma gp100 (HMB45) markers. Diseases caused by TSC mutations bear all the hallmarks of cancers including genetic mutations, evasion of growth suppression, resistance to cell death, metabolic reprogramming to avoid immune detection, and capability of invasion (3). Loss of TSC activity results in continuous activation of the mTOR pathway, which is also characteristic to various neoplasms where upstream mutations or signalling malfunction both result in mTOR activation (4). mTOR activation alters various cellular functions including cellular proliferation, autophagy, mitochondrial biogenesis, and cellular metabolism. Activation of the mTOR pathway can change anabolic cell growth processes such as protein and lipid synthesis in correlation with external growth factor or nutrient intake (5). In our previous study of LAM, apart from detecting morphological abnormalities in mitochondria and suppression of ROS production, we identified downregulation of the proliferation suppressor nuclear receptors of the retinoic acid gene family both retinoic acid receptor (RAR) and retinoid x receptor (RXR) as well as several miRNA-s that regulate RAR expression including miR29b (6). RAR and RXR are receptors of retinoic acid (RA), a metabolite of vitamin A (7). Both classes of nuclear receptors have three subtypes (α, β, and γ) and in-patient derived LAM cell lines RARβ mRNA expression was found significantly reduced (6). Interestingly, RARβ is the receptor that is associated with the anti-tumour effects of RA (8–10). In many neoplastic diseases, expression of RARβ is often downregulated or lost indicating that RARβ plays an important role in tumour suppression (11). RA is a metabolite of the lipophilic vitamin A (retinol) which is obtained from plant or animal sources in the form of carotenoids and retinyl esters, respectively (12). RA is a lipophilic molecule with three isoforms: all-trans, 9-cis and 13-cis RA and is stored in forms of retinyl esters primarily in the liver as well as the kidneys, lungs and the bone marrow (12, 13). In circulation, retinol is bound to retinol-binding protein (RBP) which enters the cells through RBP receptors (STRA6) (12). In the cells, retinol-dehydrogenase (RDH) or alcohol-dehydrogenase (ADH) oxidize retinol to retinal which is irreversibly converted to RA by the aldehyde dehydrogenase (ALDH) family also known as retinaldehyde dehydrogenase (RALDH) (12). RA binds to cellular retinoic acid-binding protein (CRABP) in the cell that carries RA into the nucleus where it binds to nuclear RARs to function as transcription factors. RA signalling is dependent upon its nuclear availability, controlled among others by RBP1, which is the carrier protein involved in the transport of retinol from the storage site to peripheral tissues (12). Interestingly, the administration of RA not only activates the transcription factor RARβ but also increases its expression (14, 15). As currently the only FDA approved drug to treat angiomyolipoma, tuberous sclerosis or LAM is rapamycin, search for additional therapeutic targets is important. Especially so, as rapamycin can only slow down disease progression and cannot offer a cure. Additionally, rapamycin has significant side effects therefore not all patients can tolerate the treatment (16, 17). Discontinuation of rapamycin, however, leads to rapid disease progression (17). Tissues affected by TSC mutation are low in RARβ expression (18, 19). As, RARβ is regulated by RA (6, 19–21), a product of vitamin A metabolism, we theorised that not just RARβ expression is low in LAM, but it is likely that enzymes of vitamin A metabolism malfunction. To test the theory vitamin A metabolic enzyme expression and activity was tested. Simultaneously, the effect of the metabolic product RA was assessed in cell migration and proliferation alone or in combination with rapamycin using TSC mutant cell lines.

Based on our results, such combination of rapamycin with RA might offer a novel therapeutic strategy if our in vitro data could be confirmed in a clinical study.

To investigate the involvement of the enzyme cascades associated with vitamin A metabolism in TSC deficient cells, human enzyme profiler arrays (RT2 PCR) were used to compare mRNA levels of specific enzymes in the human kidney angiomyolipoma cell line S102 (TSC2-/-) and its control S103 (TSC2+/+). Out of the alcohol dehydrogenase family, four enzymes (ADH1A, ADH1B, ADH1C and ADH6) were significantly upregulated and one enzyme was downregulated (ADH4) in the mutant cell line ( Figure 1A ). In the aldehyde dehydrogenase family three enzymes (ALDH1A2, ALDH1A3, and ALDH3A1) were upregulated, while five enzymes were downregulated (ALDH1A1, ALDH3B1, ALDH3B2, ALDH4A1 and ALDH5A1) ( Figure 1A ). Additionally, analysis of the array data showed significant increase in CYP2E1 mRNA level ( Figure 1A , S. Table 2 ). To predict the connection (expression, physical interaction, co-localization, etc) amongst the above described enzymes and RA in TSC mutant cells, a linear regression-based prediction algorithm analysis was performed (GeneMANIA database) (29) ( Figure 1B , S. Table 3 ). ADHs and ALDHs -especially ADH4, ALDH1A2 and ALDH1A3- were predicted to physically interact with molecules involved in the RA metabolic process and RARβ binding ( Figure 1B ). To determine whether ADHs and ALDHs are present in TSC2-deficient LAM cells, ADHs and ALDHs mRNA expression levels were quantified by qRT-PCR in four patient derived LAM cell lines and normal individual primary human bronchial smooth muscle cell (BSMC) as well as primary normal human lung fibroblast cells (NHLF) as controls ( S. Figure 1 ). Just as in the TSC2-/- angiomyolipoma cell line S102, in the primary LAM lung tissue derived cell lines the expression of ADH1, ADH4 and ALDH1A1-2-3 showed the same pattern ( Figure 1C ). Apart from vitamin A metabolism, the importance of ALDH and ADH were demonstrated in cancer cell proliferation, motility and metastasis (30, 31), due to their specific role in affecting mTOR dependent signalling. In a previous study it has been revealed that ALDH1A3 downregulation directly affects mTOR expression and its downstream signals via S6K (32). Result that ALDH1A3 mRNA was significantly upregulated in TSC2-/- S102 and patient derived LAM lung cell lines that leads to mTOR activation and downregulation of RARβ expression (6) was confirmed by immunofluorescent staining ( Figure 1D ) and western blotting ( Figure 1E ). To confirm the cell line data, primary normal and LAM lung tissue sections (n=6) were stained for RARβ protein by immunohistochemistry ( Figure 1F , S. Figure 2 ). The staining of primary LAM tissues confirmed that reduced expression of RARβ expression in the structural cells of the lung tissue is a feature of the TSC mutant LAM lungs ( Figure 1F ).

As RA is known to upregulate RARβ expression (14, 15), we set out to investigate whether RA could restore normal levels of RARβ in TSC2-/- cell lines. Four patient derived LAM lung cell lines were treated with 1 μM or 2 μM (33, 34) RA for 24 h, then RARβ expression was quantified using qRT-PCR ( Figure 2A ) and immunofluorescent staining ( Figures 2B, C ). Following incubation with 2 μM RA, RARβ mRNA ( Figure 2A ) as well as protein expression ( Figures 2B, C ) was restored to normal levels. As patients with LAM disease are treated with rapamycin, and rapamycin is known to downregulate RARβ, RARβ protein expression levels were quantified in patient derived angiomyolipoma and LAM lung cell lines after 10 nM rapamycin treatment in the presence or absence of 2 μM RA ( Figures 2D–G ). While 10 nM rapamycin mono treatment had no effect on RARβ levels, 2 μM RA increased RARβ expression even in combination with rapamycin in the TSC2-/- cell lines ( Figures 2D–G ). The effects of the above treatments were tested on mTOR activity in the angiomyolipoma cell line S102 and its control S103 by western blotting of S6 and pS6 proteins ( Figure 2F ). While 10 nM rapamycin significantly reduced S6 phosphorylation close to control levels ( Figures 2F, G ), pS6 levels in the presence of 2 μM RA mono treatment was not affected and remained just as high as in the untreated TSC-/- control. Combination treatment with 10 nM rapamycin and 2 μM RA resulted in middle ground. Significantly increased but not fully enhanced RARβ protein expression and significantly reduced pS6 levels but not as low as in the presence of 10 nM rapamycin mono treatment ( Figures 2D–G ).

Based on the data TSC mutation affects downstream signals including the vitamin A metabolic enzyme signalling cascades ( Figure 3A ). As the FDA approved rapamycin and RA in combination restored RARβ and pS6 levels we also tested both drugs on vitamin A metabolic enzyme expression and activity. Mono treatment with 2 μM RA normalised mRNA expression levels of ADH (1A, 4) and ALDH (1A1, 1A2, 1A3) ( Figure 3B ). Also, in RA treated TSC mutant S102 and primary LAM lung derived cell lines ADH and ALDH enzymes activity showed significant decrease compared to untreated controls ( Figure 3B ). Expression levels ( Figure 3B ) and enzymatic activity ( Figures 3C–F ) of metabolic enzymes were also quantified after 10 and 20 nM rapamycin and/or 2 μM RA treatments. The cultures were assessed after 24 h incubation. While in mono treatment the 20 nM rapamycin was the most efficient in reducing ALDH (35) and ADH activity, combination treatment of 10 nM rapamycin and 2 μM RA stabilised mRNA expression and activity of ADH and ALDH the most closely to TSC2+/+ control levels ( Figures 3B–F ).

To test whether normalization of enzyme levels and activity in vitamin A metabolism and suppression of mTOR activity would have an effect on cellular proliferation and migration, the effect of 2 μM RA in combination with 10 nM rapamycin were tested in mono and combination treatment in a scratch and a BrdU assay (36, 37). The combined treatment of TSC2-/- cell lines with rapamycin (10 nM) and RA (2 μM) decreased cellular migration significantly ( Figures 4A, B ). Furthermore, the combination significantly decreased the proliferation capacity detected in BrdU assay compared to rapamycin mono treatment ( Figures 4C, D ).

Apart from increased cell migration and proliferation, TSC-deficiency is characterized by structural changes of the affected tissues. Such changes cannot be detected in traditional 2D cell cultures, therefore a 3D tissue aggregates were used (27). The 3D aggregate tissues containing patient derived LAM lung cell lines developed empty sac formations after 24 h incubation which feature was reduced after 10 nM rapamycin and/or 2 μM RA treatment making the LAM cell containing co-cultures structurally similar to the aggregate cultures containing only TSC+/+ BSMC and NHLF cells ( Figure 4E ). Additionally, 10 nM rapamycin and/or 2 μM RA treatments increased RARβ protein expression even in the 3D tissue structures containing TSC mutant cell lines ( S. Figure 3 ).

In our previous study of LAM, we detected correlation between mTOR activation, mitochondrial dysfunction and downregulation of the proliferation suppressor nuclear receptor family of RAR and RXR (6). Recent studies have also shown that rapamycin treatment induced upregulation of miR-29b in LAM affected cell growth, migration, and invasion via regulation of RARβ activity (19).

In the present study, we confirmed that downregulation of RARβ is not just a feature of TSC-deficient cell lines (angiomyolipoma, LAM primary cell lines) but it is characteristically present in primary LAM lung tissue sections ( Figures 1D–F ). In many cancers (38) the activity of RARβ itself is suppressed via various pathways leading up to mTOR activation (19, 39). As RARβ levels are strongly associated with alterations in the vitamin A metabolic pathway (11), clear understanding of vitamin A metabolism in connection with TSC mutation is important for better disease control.

In the present study, we used TSC mutant angiomyolipoma and primary LAM lung derived cell lines pre-dating the rapamycin era to investigate enzyme expression and activity responsible for retinol metabolism. Based on our study the ability to metabolise retinol is seriously compromised in TSC-deficient cells ( Figure 1 ). Many enzymes, including ADH1A, ADH1B, ADH1C ADH6, ALDH1A2, ALDH1A3, and ALDH3A1 were drastically upregulated, while others including ADH4, ALDH1A1, ALDH3B1, ALDH3B2, ALDH4A1 and ALDH5A1 were significantly downregulated ( Figure 1 ). The characteristic function of ALDHs is to oxidize aldehydes that would otherwise participate in signalling pathways to induce cellular, to minimize ROS production and to mediate RA signalling cascades (40). Other studies have also shown that in diseases caused by TSC mutation which increases mTORC1 activity is associated with deregulation of ALDH expression and activity, resistance to oxidative stress, greater proliferation, migration, and invasion as well as higher levels of VEGF expression (32, 35). These regulatory mechanisms are important in regulation of proliferation, tumorigenesis and resistance to therapy (41). Expression of ALDHs are also regulated by RA compounds including the chemotherapeutic vitamin A or chemically related molecules (retinoids) as well as oncogenic pathways including the WNT/β-catenin and the MUC1-C/ERK pathways (40). CYP2E1 degrades retinoic acid (RA) and retinol to polar metabolites with toxic and apoptotic properties (42). Imbalance in the level of alcohol dehydrogenases acts as a competitive inhibitor of retinol oxidation in the liver which may reduce the biosynthesis of retinoic acid (43). It is especially important that co-expression and physical interactions amongst the metabolic enzymes of retinol are tightly controlled and important in regulation of cellular differentiation, proliferation, and migration.

Our experiments have highlighted that patients might benefit from combination of RA with the routinely used treatment of rapamycin in diseases affected by TSC mutation. In such treatment, reduction of rapamycin dosage and closely normalised vitamin A metabolic enzyme activities ( Figure 3 ) could lead to beneficial physiological effects including reduced cellular proliferation and migration ( Figure 4 ).

Importantly, several enzymes of the vitamin A metabolism and the nuclear receptor RARβ can become potential therapeutic targets in TSC mutant or deregulated neoplasms (44, 45). RA for example is an FDA approved drug for acute myeloid leukaemia as RA can normalize RARβ levels and limit cancer cell migration and consequent disease progression (45). Based on our study we propose that clinical assessment of the combination of RA with reduced dosage of rapamycin might limit adverse reactions to rapamycin in rapamycin sensitive tuberous sclerosis, LAM and angiomyolipoma patients. With reduction of rapamycin levels, the inhibitory effect of rapamycin on ALDH can also become limited which allows better balance in vitamin A metabolism and consequently in RARβ activity. Increased expression and activity of RARβ might also lead to inhibition of cellular migration, proliferation and as a result improved disease control. Further studies of improved drug concentrations and clinical assessment of our in vitro results are certainly required to modify treatment strategies for patients suffering from diseases affected by TSC mutations.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The study was approved by the Medical Research Council of Hungary (54034-4/2018/EKU). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

EA and JB-B: performed the experiments, isolated RNA and protein from NHLF, SMC and LAM, cellular staining, embedding of samples for microscopy, performed data analysis, and prepared figures. VK: generated the LAM cell lines and performed experiments on angiomyolipoma cell lines. JM, JF, TH, and GS: selected the samples. JP designed the studies. EA and JP have written the manuscript. All authors contributed to the article and approved the submitted version.

JP: TUDFO/51757-1/2019-ITM; 2020-4.1.1-TKP2020, GINOP 2.3.2-15-2016-00022; EFOP-3.6.1-16-2016-00004; and GINOP-2.3.3-15-2016-00012 HECRIN funds.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.