Towards understanding biology of leydiogioma. G protein-coupled receptor and peroxisome proliferator-activated receptor crosstalk regulates lipid metabolism and steroidogenesis in Leydig cell tumors

Leydig cell tumors (LCT) are the most common type of testicular sex cord-stromal tumor. In this report, we implicate the G-coupled estrogen receptor (GPER) and peroxisome proliferator receptor (PPAR) in regulation of lipid homeostasis and the expression of steroidogenesis-controlling molecules in clinical specimens of LCTs and cell line (mouse tumor Leydig cells; MA-10). We also show the general structure and morphology of human LCTs with the use of scanning electron microscopy and light microscopy, respectively. In LCTs, protein immunoblotting and immunohistochemical analysis revealed increased expression of GPER and decreased expression of PPARα, β and γ. Concomitantly, changes in expression pattern of the lutropin receptor (LHR), protein kinase A (PKA), perilipin (PLIN), hormone sensitive lipase (HSL), steroidogenic acute regulatory protein (StAR), translocator protein (TSPO), HMG-CoA synthase (HMGCA), and HMG-CoA reductase (HMGCR) were observed. Using MA-10 cells treated with GPER and PPAR antagonists (alone and in combination), we demonstrated there is a GPER-PPAR mediated control of cholesterol concentration. In addition, GPER-PPARα regulated estradiol secretion, while GPER-PPARγ affected cGMP concentration. It is assumed that GPER and PPAR can be altered in LCT, resulting in a perturbed lipid balance and steroidogenesis. In LCTs, the phosphatidylinositol-3-kinase (PI3K)-Akt-mTOR signaling pathway was disturbed. Thus, PI3K-Akt-mTOR, together with cGMP, can play a role in LCT proliferation, growth, and metastasis as well as lipid balance control. In conclusion, we discuss the implications of GPER-PPAR interaction with lipid metabolism and steroidogenesis controlling-molecules in LCT biology that can be used in future studies as potential targets of diagnostic and therapeutic implementations.


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It is worth noting that biosynthesis of sex steroids is multi-level, controlled process [49]. It 149 requires the coordinated expression of number of genes, proteins of various function 150 (receptors e.g. lutropin receptor; LHR, enzymes, transporters e.g. translocator protein; TSPO, 151 steroidogenic acute regulatory protein; StAR, and regulators), signaling molecules (e.g.  This study aims to determine the potential link between GPER and PPAR and whether   Twenty-four hours before the experiments, the medium was removed and replaced with a 189 medium without phenol red supplemented with 5% dextran-coated, charcoal-treated FBS (5%  To calculate the amplification efficiency serial cDNA dilution curves were produced for all 237 genes. A graph of threshold cycle (Ct) versus log10 relative copy number of the sample from a 238 dilution series was produced. The slope of the curve was used to determine the amplification 239 efficiency: %E = (10 −1/slope -1) × 100. All PCR assays displayed efficiency between 94% and 240 104%.   Then, blots were blocked with 5% nonfat dry milk in TBS, 0.1% Tween 20, overnight at 4 °C 265 with shaking, followed by an incubation with respective antibodies ( Table 1). The membranes 266 were washed and incubated with a secondary antibody conjugated with the horseradish-

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The relative protein levels were expressed as arbitrary units.   305 The production of cGMP in control and treated with GPER and PPAR (alone or in  The sensitivity of the assay was 10.6 pg/mL. The absorbance (λ = 450 nm) was measured.

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Data were expressed as mean ± SD.

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The measurements were performed with the use ELISA apparatus (Labtech LT-4500). Three biological repeats of each sample (n = 7) and three independent experiments were 318 performed. Each variable was tested using the Shapiro-Wilk W-test for normality. The    the interstitial space ( Fig.2 A, B). In LCTs, most cells possessed a large polygonal shape with abundant cytoplasm, indistinct cell borders, and regular round to oval nuclei. The nucleus was 342 found to be frequently prominent (Fig. 2b). Occasionally, cells as those noted above were 343 found to possess distinct cell borders and smaller nuclei (Fig. 2b'). Small cells with scant, 344 densely eosinophilic cytoplasm and a grooved nuclei (Fig. 2b") and spindle-shaped 345 (sarcomatoid) cells (Fig. 2b"') were observed as well.

Expression and localization of GPER and PPARs in LCTs
In LCTs, increased expression of GPER (p < 0.05) and decreased expression of PPARα 350 (p < 0.001), PPARβ (p < 0.01), and PPARγ (p < 0.001) was seen when compared to controls 351 (Fig.3A, B). Corresponding to GPER and PPARs protein expression changes their mRNA 352 expressions in LCTs are presented as supplementary material (Fig.3'). 353 No changes in GPER localization and staining intensity was found in control Leydig cells 354 and LCTs (Fig.4 A, A'). Specifically, the staining was exclusively cytoplasmic and of  HMGCR was observed when compared to normal Leydig cells (Fig. 5A, B). The expression 368 of LHR and PKA was increased (p < 0.05 and p < 0.01, respectively) as well as that of 369 HMGCS and HMGCR (p < 0.001 and p < 0.05, respectively). In contrast, PLIN and StAR are presented as supplementary material (Fig.5').

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In control Leydig cells and LCTs, cytoplasmic expression of LHR was found (Fig.6 A, A').

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The immunostaining was of moderate intensity in control Leydig cells but was weak and 376 present in minority of cells of LCTs. No differences were found in PKA distribution and 377 immunostaining (Fig. 6 B, B'), with strong staining present in control and tumor Leydig cell 378 cytoplasm. PLIN distribution was cytoplasmic in control Leydig cells and LCTs (Fig. 6 C, 379 C'). In control Leydig cells, staining intensity was strong while found to be weak in LCTs.

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Increased HSL staining intensity was found in LCTs when compared to control cells (Fig. 6D,

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In LCTs, PI3K and Akt expression was increased (p < 0.05,) while no observable changes 395 in mTOR expression was found when compared to controls (Fig. 5A, B).  In the present study, we examined the cellular organization and molecular mechanisms, 413 including GPER and PPAR signaling, lipid balance, and steroidogenesis-regulating molecular 414 interactions that regulate LCT biology.   was reported [87]. Moreover, in patients with testicular cancer, hCG treatment caused excess 535 of estradiol secretion by the tumor [88]. In a mouse tumor Leydig cell line (mLTC-1), 536 epidermal growth factor increased StAR activity and steroid production efficiency in a time-537 and dose-dependent manner with involvement of ERK, while LHR expression was 538 significantly reduced [88]. In mice (C57BL/6J) with LCTs, cessation of steroidogenesis was 539 present when LH and cAMP were removed [89]. We found prominent changes in LHR and It is worth adding that lipids have been recognized as a component of metabolic 565 reprogramming in tumor cells [98]. Many tumors show a reactivation of de novo fatty-acid 566 synthesis for generation of membrane structural lipids; thus, they do not rely on lipids from 567 the bloodstream [99]. Modulated lipid synthesis may also have a non-cell-autonomous role in 568 cancer development. The growth and metastasis promotion of LCT cannot be excluded with 569 participation of adipocytes [100]. In addition to their structural and signal transduction 570 roles, lipids can also be broken down into bioactive lipid mediators, regulating a variety of 571 carcinogenic processes including cell growth, cell migration, and metastasis formation, as 572 well as the uptake of chemotherapeutic drugs [101,102]. Therefore, based on our results activation of mTOR is possible by other signaling pathway or can be related to advanced LCT 590 development [107]. Our earlier studies in mouse tumor Leydig cells revealed that GPER and 591 PPAR inhibition activate PI3K and Akt [52] but mTOR is modulated diversely; inhibits by 592 GPER antagonist alone and together with PPARγ antagonist as well as activates by GPER 593 with PPARα together and the latter alone (supplementary Fig.7').