Perirenal White Adipose Tissue Browning and Epithelial-Mesenchymal Transition, Contributes To Tumor Development in Kidney Cancer

Tumor cells can interact with neighboring adipose tissue and adipocyte dedifferentiation appears to be an important aspect of tumorigenesis. We evaluated the size of adipocytes in human adipose explants from normal (hRAN) and kidney cancer (hRAT); changes in the expression of WAT and BAT/beige markers in hRAN and hRAT; and changes in the expression of epithelial-mesenchymal transition (EMT) cell markers; in human kidney tumor (786-O, ACHN and Caki-1) and non-tumor (HK-2) epithelial cell lines incubated with the conditioned media (CMs) of hRAN and hRAT. We observed that hRAT adipocytes showed a signicantly minor size compared to hRAN adipocytes. Also, we observed that both Prdm16 and Tbx1 mRNA and the expression of UCP1, TBX1, PPARγ, PCG1α, c/EBPα LAP and c/EBPα LIP was signicantly higher in hRAT than hRAN. Finally, we found an increase in vimentin and N-cadherin expression in HK-2 cell incubated for 24 h with hRAT-CMs compared to hRAN- and control-CMs. Furthermore, desmin and N-cadherin expression also increased signicantly in 786-O when these cells were incubated with hRAT-CMs compared to the value observed with hRAN- and control-CMs. The results obtained, together with the results previously published by our group, allow us to conclude that perirenal white adipose tissue browning and epithelial-mesenchymal transition, contributes to tumor development in kidney cancer.


Introduction
In recent years it has been shown that tumor progression also depends on the bidirectional dialogue between tumor epithelial cells and surrounding stromal cells. Among the different types of cells that share microenvironment with renal epithelial cells, renal adipose tissue is one of the most abundant. Adipose tissue (AT) is mainly responsible for the depository and delivery of energy in response to systemic demands but also is currently recognized as an endocrine and immunomodulating organ that contributes to human physiology, through both systemic and local-speci c functions [1]. Cancer cells and cancer associated adipocytes (CAAs) interact with each other, which promotes the formation of a unique microenvironment and stimulates tumor growth and metastasis [2][3][4][5]. Therefore, the role of AT on tumor metabolism is becoming increasingly important [6,7].
Renal cell carcinoma (RCC) is one of the ten most often diagnosed cancers worldwide. Each year over 300000 new RCC cases are diagnosed and nearly 140000 patients die of this disease [8]. RCC is a highly metastatic cancer; nearly 30% of all RCC patients have developed metastasis at the time of diagnosis [9]. Clear cell renal cancer (ccRCC) is the most frequent and aggressive type of RCC, and usually represents 80-85% of all RCC. Furthermore, the incidence of RCC has increased globally by 2-3% per year [10].
Adipocyte dedifferentiation appears to be an important aspect of tumorigenesis [11]. Recent work on breast tissue has shown that the adipocytes that surround tumor epithelial cells undergo a more rapid depletion of their lipid stores than those that are further away from the tumor [12]; and the adipocytes on the invasive front are smaller than those observed at a distance, implying the existence of lipolysis [13;3]. Recently we observed that human perirenal AT fragments from kidney tumor patients (hRAT) expressed signi cantly higher amounts of the leptin and ObR (leptin receptor) than human perirenal AT fragments from living kidney donors (hRAN) [5]. In addition, leptin can act locally by increasing lipolysis accompanied by a decrease in the size of adipocytes. Therefore, in this work we evaluated the size of adipocytes of hRAN and hRAT.
Two main types of adipose tissue have been described, namely white adipose tissue (WAT) with a classical energy storage function, and brown adipose tissue (BAT) with thermogenic activity [7]. In recent decades, a phenomenon known as 'browning' of WAT has been described, which was rst reported by Young and colleagues in 1984 [14]. This process is triggered by the increased gene expression levels of different markers involved in the BAT adipogenic differentiation, including PPARG or PGC-1 [15]. The white adipocytes that undergo the browning process are called beige adipocytes. The beige cells of white adipose tissue, as occurs with brown adipocytes, are identi ed by their multilocular morphology, the high number of mitochondria, and the expression of a set of genes speci c to brown fat such as UCP1, PGC1α, and PRDM16. Although brown and beige cells are morphologically similar and both have the ability to perform thermogenesis, they have different origins and responses to certain stimuli [16]. In recent years, a possible browning process of peritumoral mammary adipose tissue in breast cancer has been postulated. Both due to phenotypic modi cations of the peritumoral adipose tissue, and due to changes in the expression of genes involved in the browning process [17-19;7;20]. Gantov et al. recently demonstrated that beige adipocytes favor tumor progression of mouse mammary epithelial cell lines, both tumor and non-tumor [20]. Our group recently demonstrated that renal peritumoral AT (hRAT) favors tumor progression through soluble factors that it secretes into the microenvironment, unlike normal renal adipose tissue (hRAN). Thus, hRAT would be able to stimulate a protumorigenic behavior of human renal epithelial cells, both tumor and nontumor. [5]. Tumor-induced differentiation to beige/brown adipose tissue is an important contribution to the hypermetabolic state of breast cancer [20]. Therefore, we evaluated changes in the expression of WAT and BAT/beige markers in hRAN and hRAT by different methods.
The extracellular matrix (ECM) is a complex structure made up of different proteins, proteoglycans and polysaccharides. Endocrine activity of adipocytes includes several factors implicated in ECM formation and remodeling [21]. We recently observed that conditioned media (MCs) of human adipose explants from kidney cancer (hRAT) increased the metastatic capacity of tumor and non-tumor human renal epithelial cells, unlike hRAN-CMs [5]. The epithelial-mesenchymal transition (EMT) is a characteristic process of epithelial cells when they acquire migratory capacity. Therefore, in this work we decided to evaluate changes in the expression of EMT cell markers characterized by the expression of mesenchymal markers vimentin, desmin and N-cadherin; in human kidney tumor (786-O, ACHN and Caki-1) and non-tumor (HK-2) epithelial cell lines incubated with the CMs of hRAN and hRAT.

Results
The adipocytes that surround the kidney tumor are smaller than the adipocytes that surround a normal kidney.
We evaluate the size of adipocytes from different renal adipose tissues. Speci cally, we compared the normal kidney adipose tissue (hRAN) and renal peritumoral adipose tissue (hRAT). We observed signi cant changes in the size of adipocytes in response to the presence of the tumor. The hRAT adipocytes showed a signi cantly smaller size compared to the hRAN adipocytes ( Fig. 1). This change in adipocyte size, together with the increased expression of leptin and its receptor in hRAT vs. hRAN [5], suggest an increase of lipolysis by hRAT adipocytes compared to hRAN adipocytes.
hRAT showed an increase in the gene expression of Prdm16 and TBX1 compared to hRAN.
To evaluate the browning of the perirenal AT, we measured mRNA levels of Prdm16, TBX1, Ucp1 and PGC1 alpha in AT from normal and tumor kidney. Increased levels of Prdm16 and TBX1 mRNA in hRAT compared to hRAN (Fig. 2, p<0.05) were found. Although no signi cant differences were found in Ucp1 and PGC1 alpha mRNA, a tendency to increase was seen in hRAT-CMs (Fig. 2). UCP1 and PGC1 alpha expression was increased in hRAT adipocytes compared to hRAN.
We performed immunohistochemistry assays on adipose tissue from normal and kidney cancer to measure UCP1, PGC1 alpha and HSP protein levels and localization. UCP1 and PGC1 alpha expression levels increased in hRAT adipocytes compared to hRAN adipocytes (Fig. 3, p<0.05).

Soluble factors secreted by hRAT stimulate the expression of EMT cells markers in tumor and non-tumor human kidney cells.
We decided to evaluate changes in the expression of EMT cell markers; in human kidney tumor (786-O, ACHN and Caki-1) and non-tumor (HK-2) epithelial cell lines incubated with the conditioned media (CMs) of human adipose explants from normal (hRAN) and kidney cancer (hRAT) tissue. We found an increase in vimentin and N-cadherin expression in HK-2 cell incubated for 24 h with hRAT-CMs compared to hRAN-and control-CMs (p<0.05) ( Fig. 5A and I). The same tendency was observed for the expression of desmin, but it was not statistically signi cant (Fig. 5E). Furthermore, desmin and Ncadherin expression also increased signi cantly in 786-O when these cells were incubated with hRAT-CMs compared to the value observed with hRAN-and control-CMs ( Figure 5F and J, p<0.05). However, no signi cant changes in vimentin expression were observed in 786-0 cells (Fig. 5B). Likewise, we did not nd signi cant changes in the expression of vimentin, desmin or N-cadherin in the two tumor renal epithelial cell lines originating from metastatic sites (ACHN and Caki-1 cells) (

Discussion
Renal cancer (RCa) is considered to be the fth most common type of cancer worldwide, having a high mortality rate in both men and women. In Argentina, 4100 new cases are diagnosed each year in both sexes, being more frequent in men. Tumor development and maintenance of a cancerous phenotype, requires a bidirectional communication between epithelial cells and the stromal environment. Renal adipose tissue is one of the most abundant cell types surrounding renal epithelial cells. This tissue is a bioactive endocrine organ that secretes soluble factors and contributes signi cantly to the composition of the extracellular matrix. Our group has shown that the periprostatic adipose tissue of patients with prostate cancer can regulate the behavior of the tumor, both in the early and late stages of the disease [23]. Furthermore, we have worked with adipose tissue fragments from human breast tumors (hATT) and normal breast glands (hATN), from which we obtained the corresponding conditioned media (hATT-CMs and hATN-CMs). We show that hATT is capable of stimulating the growth and metastatic capacity of mammary tumors by different mechanisms, unlike hATN [24;3;25]. Recently, we demonstrated that renal peritumoral adipose tissue undergoes a process of adaptation to changes locally generated by the tumor [5]. Also, we show that this hRAT is capable of stimulating a protumorigenic behavior of renal epithelial cells. We observed that hRAT expresses signi cantly higher amounts of leptin and ObR, relative to hRAN. It has been shown that leptin has a pro-tumorigenic function and act locally by increasing lipolysis. This increase in lipolysis is accompanied by a decrease in the size of adipocytes. Along these lines, our results show a signi cant decrease in the mean size of hRAT adipocytes with respect to the size of hRAN adipocytes (Fig. 1). The increased expression of both leptin and its receptor, together with the decrease in the size of hRAT adipocytes, allow us to suggest an increase in lipolysis in hRAT adipocytes. The increase in lipolysis would allow a greater availability of energy to the tumor, favoring its development.
Adipocytes are the main stromal cells in the mammary microenvironment, and crosstalk between adipocytes and cancer cells may play a critical and important role in cancer maintenance and progression. Tumor induced differentiation to beige/brown adipose tissue is an important contribution to the hypermetabolic state of breast [20] and prostate [7] cancer, but still the bibliography says nothing about browning and kidney cancer. And, the effect of epithelial cell-beige adipocyte communication on tumor progression remains unclear. To begin to elucidate this, we evaluated the expression of different markers WAT and BAT / beige adipocytes in hRAT and hRAN. The browning of WAT is triggered by the increased gene expression levels of different markers involved in the BAT adipogenic differentiation, including PPAR gamma or PGC-1 alpha [26]. Also, PPAR gamma induces the expression of C/EBP, which makes this gene a key regulator of WAT differentiation [27]. PPAR gamma also induces the expression and production of UCP-1 in these beige adipocytes [28]. UCP1 this transmembrane protein uncouples the electron transport chain (ETC) by pumping protons from the intermembrane space back into the mitochondrial matrix, thereby generating heat rather than ATP [29]. As well, PRDM16 is a master regulator gene of brown adipocyte differentiation and TBX1 beige adipocyte marker [18]. We observed that both Prdm16 and Tbx1 genes and the protein expression of UCP1, TBX1, PPARγ, PCG1α, c/EBPα LAP and c/EBPα LIP was signi cantly higher in hRAT than hRAN (Fig. 2-4). This is the rst work that demonstrates a transdifferentiation of WAT adipocytes in the human perirenal AT that surrounds a renal tumor, since we observed an increase in the amount of beige adipose tissue in hRAT, as opposed to the hRAN. In light of these results, together with those previously described [5], we postulate that the renal tumor would be regulating the differentiation of the surrounding WAT and its browning.
The browning process might have a role in the development of kidney tumors. Nowadays, and to deepen this possible regulation, we set out to study changes in WAT and BAT/beige adipocytes markers in hRAN fragments incubated with MCs from tumor kidney cell lines. Preliminary results allow us to observe an increase in the expression of BAT/beige adipocyte markers in hRAN, which would support the hypothesis that the renal tumor would be stimulating the browning of the surrounding TA.
Finally, we evaluate the ability of hRAT-and hRAN-CMs to produce changes in the expression of EMT cell markers; in human kidney tumor and non-tumor epithelial cell lines incubated with the MCs of hRAN and hRAT. Previosuly we showed that hRAT expresses signi cantly higher amounts of leptin and ObR, relative to hRAN [5]. It has been described that leptin induces a broblastoid morphology evidenced by the decrease in the expression of epithelial markers (occludin, Ecadherin) and an increase in mesenchymal markers ( bronectin, N-cadherin, and vimentin) in breast cancer [30]. In accordance with our previous results, where we observe that hRAT-CMs increased the metastatic capacity of tumor and non-tumor human renal epithelial cells, we found an increase in vimentin and N-cadherin expression in HK-2 cell, desmin and N-cadherin expression in 786-O cells incubated with hRAT-CMs compared to hRAN-and control-CMs. Likewise, it has been described that leptin is capable of stimulating EMT. And we previously demonstrated that hRAT expresses higher amounts of leptin than hRAN. ECM remodeling in pathological processes included obesity and tumor progression, since it needs to undergo a reorganization to allow accommodation of the hypertrophic adipocytes allowing access to the lipid content of adipocytes [31]. During the tumor progression, ECM remodeling is a key factor involved that allow invasion and cell migration [32]. Endocrine activity of adipocytes includes several factors implicated in ECM formation and remodeling through switch on the synthesis and release of molecules such as collagenases and/or metalloproteinases, favoring a better environment for cell migration out of the primary tumor [7]. Therefore, we postulate that this adipokine could be involved in increasing the expression of EMT markers. It is striking to see this increase in the non-tumor cell line. This is the rst report demonstrating that human adipose tissue from renal tumor (hRAT) presents beige/brown adipocyte characteristics, unlike human adipose tissue from normal renal (hRAN). This browning process could be stimulated by the kidney tumor itself, and play an important role in renal tumor development. Furthermore, we demonstrate that hRAT produces soluble factors that stimulate the metastatic capacity of both tumor and non-tumor human kidney cells.

Reagents
Reagents were from Sigma Chemical Co (St. Louis, MO, USA), tissue culture asks, dishes, and multi-well plates were from Falcon Orange Scienti c (Graignette Business Park, Belgium), and culture media from both tissue and cell lines and supplements were from Gibco BRL (Carlsbad, CA, USA) [3][4][5].

Sample collection and handling
Human adipose tissue explants from cancerous (hRAT; n=23) kidneys were obtained from patients to whom a partial or total (tumor) nephrectomy was performed. Human adipose tissue explants from normal kidneys (hRAN; n=19), were obtained from live kidney donors who had not received previous chemotherapy or radiotherapy treatment. The median of body mass index (BMI) of patients was: 26.8 kg/m 2 for patients with renal tumor (hRAT), and 24.9 kg/m 2 for living kidney donors (hRAN). (BMI) (kg/m2) was calculated as weight (kg) divided by height (m) squared.
Samples were transported in PBS and processed immediately. On average, 2 h elapsed from the acquisition of the surgical sample until it was processed under a sterile laminar ow hood. The project was approved by the Medical School's ethics committee (Universidad Nacional de Cuyo, Argentina) according to Declaration of Helsinki to experimentation with human subjects. All patients gave their informed consent to undergo tissue harvesting for this research [5].

Gene expression by RT-qPCR analysis
Total RNA was extracted from 100mg of tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quanti ed according to its absorbance at 260nm (NanoDrop 2000, Thermo Scienti c, Wilmington, USA). Contaminating genomic DNA was degraded with DNAse RQ1 (Promega, Madison, USA), cDNA was synthesized from one microgram of total RNA using 300pmol oligo-dT primers, 10mM dNTP (Thermo Scienti c, Wilmington, USA) and 200U M-MLV reverse transcriptase (Promega, Madison, USA). Real-time PCR was performed in a nal volume of 20 uL containing 50 ng cDNA, 3mM MgCl2, PCR LightCycler-DNA Master SYBRGreen reaction mix (Roche, Indianapolis, USA) and 0.5 mM of each speci c primers (Table 1). Ampli cation was performed in a using LightCycler thermocycler (Roche, Indianapolis, USA). Controls without reverse transcription were included to ensure that ampli cations were from mRNA and not from genomic DNA. Amplicons were characterized according to their melting temperature and size. The mRNA level of each target gene was calculated using the 2ΔΔCt method and normalized against the mRNA of β-actin [4][5]. Tissues (hRAT and hRAN) were xed in 4% formaldehyde and embedded in para n. They were afterwards cut into sections of 3 m thickness with a microtome, depara nized and stained with hematoxylin-eosin (H&E). Images were taken with a Nikon Eclipse E200 Microscope tted with a digital still camera Micrometric SE Premium (Nikon Corp., Japan) at 100x magni cation. Adipocyte area quanti cation (measuring adipocyte perimeter) in the three tissue types was performed in 8-10 elds of each preparation as mentioned above [5].
Immunohistochemistry 10 µm serial cuts were performed on the same tissue samples embedded in para n used for H&E staining. UCP1, PGC1 alpha and HSP expression were studied by means of immunohistochemistry. Brie y, hRAN and hRAT microtome slides were rst depara nized, and then a heat-mediated antigen retrieval, endogenous peroxidase blocking and nonspeci c tissue blocking were performed. Slides were then incubated with the different primary antibodies at 4 °C, and after that with an anti-rabbit biotinylated secondary IgG antibody. Finally, slides were incubated with peroxidase-conjugated streptavidin. Peroxidase reaction was performed with chromogen 3,3′-diaminobenzidine (DAB) (DAKO LSAB + Kit, HRP).
Hematoxylin counter stain was performed. Serial cuts incubated in the absence of primary antibody were used as negative controls. Images were taken with a Nikon Eclipse E200 Microscope tted with a Micrometric SE Premium (Nikon Corp., Japan) digital still camera at 100x and 400x magni cation. DAB staining quanti cation in the three tissue types was performed in 5 elds of each preparation as mentioned above [3][4][5].

Preparation of conditioned media (CMs) from hRAN and hRAT
Adipose tissues were washed with cold PBS and weighed. hRAN or hRAT were plated in culture ask with M199 culture medium (Invitrogen™; 1 g tissue/10 ml M199), supplemented with gentamicin (50 µg/ml) and incubated for 1 h at 37 ºC in 5 % CO 2 . After that, the medium was removed and replaced with fresh medium and the tissues were incubated for 24 h.
Subsequently, the supernatant was collected and ltered using lters with 0.22 µm membranes. Then, supernatants were aliquoted into 1-ml fractions and immediately stored at -80 ºC. The control-CMs were obtained from the collection of serum-free M199 medium after 24 h of incubation in a culture ask at 37 ºC in 5 % CO 2 [5]. The four cell lines were cultured in DMEM-F12 medium with 10 % FBS and 2 µg/ml insulin; and were maintained at 37 ºC in 5 % CO 2 [5].

Western Blot analysis
In order to evaluate protein expression levels, Western blots were performed. UCP1, TBX1, PPARγ, PCG1α, c/EBPα LAP, c/EBPα LIP, adiponectin, leptin, vimentin, desmin and N-cadherin were measured after incubation of the epithelial cell lines with the different CMs obtained. In order to lyse cells, Ripa buffer was used. Total protein in samples was quanti ed by Bradford method. Proteins were separated in a SDS-PAGE 12 gel, and electrotransferred to a nitrocellulose membrane (Amersham). The membrane was later blocked with bovine serum albumin (Sigma-Aldrich, 0055K) and then incubated with the different antibodies ON at 4ºC. The membranes were later washed, and incubated with proper secondary antibodies conjugated with biotione, and subsequently the signal was ampli ed with streptovidin. Antibody complexes were visualized by means of chemiluminescence (ECL; GE Helathcare). Bands were quanti ed by densitometry using FIJI Image processing package [22]. In the cell extracts, β-actin level in samples was used to determine that equal quantities of proteins were loaded in the gel [3][4][5].

Statistical methods
The statistical signi cance between different experimental conditions was evaluated by t-test or one-way ANOVA. Tukey´s post-hoc tests were performed within each individual treatment. The results are presented as mean ± SEM. Results were considered signi cant at p < 0.05. Figure 1 Microscopic evaluation of different renal adipose tissues. Slides from adipose tissue samples from normal kidney of hRAN; and tissue samples of tumor kidney attached to the tumor of hRAT. Adipose tissue fragments were cut in para n, stained with H&E, and observed under light microscope. Adipocyte size was quanti ed with Image J software (NIH). The graphic show median ± SEM of two independent experiments. *p < 0.05 hRAT attached to the tumor vs. hRAN. Magni cation: 100X.

Figure 2
Relative fold expression of Prdm16, TBX1, Ucp1 and PGC1 alpha gene expression from hRAN and hRAT. The mRNA pro les of Prdm16, TBX1, Ucp1 and PGC1 alpha from different adipose tissue were analyzed by qRT-PCR and normalized by their relative ratio to GAPDH. Data are mean ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *p<0.05.

Supplementary Files
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