Introduction: Metformin (MF) intake could be associated with a favorable outcome in sunitinib (SUT)- and axitinib (AX)-treated clear cell renal cell carcinoma (ccRCC) patients. Functionally, MF induces miR-205, a microRNA serving as a tumor suppressor in several cancers. Methods: Real-time quantitative PCR, viability assays, and Western blotting analyzed MF and SUT/AX effects in RCC4 and 786-O cells. A tetracycline-inducible overexpression model was used to study the role of miR-205 and its known target gene, VEGFA. We analyzed miR-205 and VEGFA within a public and an in-house ccRCC cohort. Human umbilical vein endothelial cell (HUVEC) sprouting assays examined miR-205 effects on angiogenesis initiation. To determine the influence of the von Hippel-Lindau tumor suppressor (VHL), we examined VHLwt reexpressing RCC4 and 786-O cells. Results: Viability assays confirmed a sensitizing effect of MF toward SUT/AX in RCC4 and 786-O cells. Overexpression of miR-205 diminished VEGFA expression – as did treatment with MF. Tumor tissue displayed a downregulation of miR-205 and an upregulation of VEGFA. Accordingly, miR-205 caused less and shorter vessel sprouts in HUVEC assays. Finally, VHLwt-expressing RCC4 and 786-O cells displayed higher miR-205 and lower VEGFA levels. Conclusion: Our results support the protective role of MF in ccRCC and offer functional insights into the clinical synergism with tyrosine kinase inhibitors.

Preclinical and epidemiological studies in several malignancies suggest a tumor-suppressive role of metformin (MF), a biguanide originally used for treating type 2 diabetes [1]. There is increasing evidence that combining MF and antineoplastic agents may improve outcomes of patients suffering from lung [2], colorectal, and prostate cancer [3]. Regarding clear cell renal cell carcinoma (ccRCC), regular MF intake was associated with a lower cancer incidence in patients with type 2 diabetes [4]. Moreover, two recent studies identified regular MF intake as prognostically favorable in ccRCC patients treated with tyrosine kinase inhibitors (TKIs) [5, 6], a drug class representing a cornerstone of ccRCC therapy by addressing angiogenesis-related pathways [7].

Functionally, MF is known for inhibiting tumor growth by activating AMP-activated protein kinase (AMPK) and counteracting mechanistic target of rapamycin (mTOR) signaling [8‒10]. Additionally, MF can alter microRNA (miR) expression – thereby functioning as an antitumor agent [11, 12]. In specific, MF treatment caused a downregulation of miR-21 in ccRCC cells [13]. In contrast, MF induced miR-205 expression in murine embryonic fibroblasts [14]. This miR is frequently downregulated in cancer, e.g., prostate cancer [15] and ccRCC [16]. Additionally, miR-205 was shown to specifically target VEGFA in malignancies such as breast cancer [17] and ccRCC [16]. We therefore investigated whether MF administration influenced miR-205 expression in ccRCC cells – thereby potentially regulating VEGFA expression and TKI response in vitro.

Given that a functional loss of the von Hippel-Lindau gene (VHL) is a frequent event in ccRCC pathogenesis and given its role in angiogenesis regulation [18, 19], we finally investigated the influence of the VHL status on the miR-205/VEGFA axis.

Cell Culture and Reagents

Cell lines were obtained from ATCC (American Type Culture Collection, Chicago, IL, USA): HEK293T (ATCC cat# 3216), 786-O (ATCC cat# 1932, VHLmut), and RCC4 (ATCC cat# 11268, VHLmut). Cells were cultured in DMEM high glucose (4.5 g/L), supplemented with 10% fetal calf serum, 2 mmol/L glutamine, and 100 U/mL penicillin/streptomycin (all PAA, Pasching, Austria). HK-2 (ATCC cat# 2190, immortalized human renal proximal tubule epithelial cells) were cultured in keratinocyte serum-free medium supplemented with 0.05 mg/mL bovine pituitary extract, 5 ng/mL human recombinant epidermal growth factor, and 100 U/mL Penicillin/Streptomycin. MF was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and diluted in phosphate-buffered saline. Sunitinib (SUT) and axitinib (AX) were kindly provided by Pfizer (New York, NY, USA) and diluted in DMSO.

Patients and Tissue Sample Preparation

Tumor tissue and adjacent benign tissue of n = 32 RCC patients undergoing radical nephrectomy between 2006 and 2010 at the Department of Urology and Pediatric Urology, University Hospital Würzburg, were included in this study. Of note, we did not perform microdissections. Instead, an experienced pathologist macroscopically identified malignant and nonmalignant tissue and confirmed the presence of >90% malignant cell content. Specimens were frozen in liquid nitrogen immediately after resection. Storage was at −80°C until RNA extraction was performed as described below.

Tetracycline-Inducible miR-205 Model in ccRCC Cells

To overexpress miR-205 in ccRCC cells, we used a tetracycline-inducible (Tet-On) model described before [20]. In brief, we cloned the parental miR-205 stem-loop sequence into the lentiviral LT3GEPIR vector (Addgene#11177) using the EcoRI and XhoI restriction sites. Lentivirus was then produced in HEK293T cells using standard protocol, and 786-O and RCC4 cells were subsequently infected with LT3GEPIR lentiviral particles bearing the miR-205 core sequence. Cells were selected using 2 μg/mL puromycin for stable clones 2 weeks prior to experimentation.

Stable Transduction of Wild-Type VHL in ccRCC Cells

786-O and RCC4 cells stably expressing VHLwt, and empty vector (EV) controls were a kind gift from Beatrice Griffiths (Institute of Cancer Research, London, UK). In brief, both cell lines were stably transduced using pBabe puro HA-VHLwt (Addgene# 19234) to express HA-tagged VHLwt or pBabe puro (Addgene# 1764) serving as EV controls.

Induction of miR-205 Expression and Drug Treatment

Cells were plated in a final concentration of 1 × 104 to 5 × 104 cells/well in 96-well plates and 1 × 105 to 3 × 105 in 6-well plates. For miR-205 induction, either 1 μg/mL doxycycline (purchased from Sigma-Aldrich, Saint Louis, MS, USA, dissolved in 100% EtOH) or 1 μL/mL 100% EtOH was applied to 786-O and RCC4 cells transduced with LT3GEPIR miR-205. Induction was verified by observation of GFP (green fluorescent protein) expression under a phase-contrast microscope and subsequent real-time quantitative PCR (qRT-PCR). Protein and RNA isolation was carried out 72 h after induction. MF/SUT/AX was added 24 h after plating. Online suppl. Table S1 (for all online suppl. material, see https://doi.org/10.1159/000535025) summarizes compounds and incubation times for each experiment.

Cell Viability Assay

Cells were analyzed with MTS CellTiter96 Proliferation Assay (Promega, Madison, WI, USA) at 490 nm with a monochromator (Bio-Rad, Hercules, FL, USA) according to the manufacturer.

RNA Extraction and Reverse Transcription

Total RNA was extracted from cells after miR-205 induction and/or drug treatment as well as from patient samples using phenol-chloroform extraction (TRIzol agent, Life Technologies, Carlsbad, MN, USA). Total RNA concentration was determined with a bioanalyzer (Agilent, Santa Clara, CA, USA). cDNA was synthesized from total RNA with stem-loop reverse transcription primers according to the TaqMan miR assay protocol for microRNA expression and according to the IMPROM-II reverse transcription kit protocol (Promega, Madison, WI, USA).

Real-Time Quantitative PCR

Mature miR expression was quantified in tissue samples with TaqMan miR assay kits and an Applied Biosystems 7.900HT system. We followed the manufacturer’s protocol (Life Technologies, Carlsbad, MN, USA). Expression of snRNA RNU6B served as normalization. Relative miR expression was calculated with the comparative ∆Ct method (∆Ct sample = Ct sample - Ct RNU6b). Fold changes in miR expression were determined by the 2−∆∆Ct method. mRNA analysis of VEGFA expression was performed according to standard procedures. Expression of β-Actin was used for normalization. All primer sequences are available upon request, if not bought as ready-to-use kits. Mean Ct was determined from triplicate PCRs.

Western Blot Analysis

Cells were lysed in PhosphoSafe (MERCK Life Sciences, Billerica, MA, USA). Equal amounts of protein isolates were separated on SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). For VEGFA protein expression, we used 1 mg/mL polyclonal antibodies (Abcam, Cambridge, UK). Vimentin (Sigma-Aldrich, Saint Louis, MS, USA) served as loading control. For visualization, we used horseradish peroxidase-coupled secondary antibodies (Abcam and Agilent) and the ECL Plus kit (GE Healthcare, Chalfont St. Giles, UK). iBright FL1000 (Thermo Fisher, Waltham, MS, USA) was used for processing and visualization. All western blots were repeated three times showing comparable results.

Endothelial Sprouting Assay

Assays were performed as described previously [21]. Briefly, human umbilical vein endothelial cells (HUVECs) (PromoCell, C-12205) were cultured overnight in hanging drops in M200 medium with 25% methylcellulose (Sigma, M0512) to form spheroids. Then, spheroids were retrieved and centrifuged at 300 g for 5 min. They were embedded into a collagen matrix in a 24-well plate consisting of 43% collagen (3.5 mg/mL, Corning/OMNILAB GmbH & Co. KG # 354236), 41% M200 medium with 25% methylcellulose and 40% fetal calf serum, 15% NaHCO3 (15.6 mg/mL), and 1% 1 m NaOH. Spheroids were incubated for 30 min at 37°C for polymerization of the collagen matrix. Then, medium was added, and cells were incubated for 24 h. Afterward, spheroids were fixed with 4% paraformaldehyde for 20 min at room temperature and washed with PBS. Pictures were taken with a bright-field microscope and analyzed using ImageJ.

Statistical Analysis

Statistical analysis was performed using R 3.10 (http://www.r-project.org), and figures were drawn with GraphPad Prism 7. Pearson’s χ2 test was used to realize intergroup comparison. If two means of continuous data were compared, a two-sided unpaired Student’s t test was used. miR-205-5p and VEGFA mRNA expression data from the TCGA KIRC cohort were accessed via ENCORI (https://starbase.sysu.edu.cn) [22].

Decreased Viabilities of RCC4 and 786-O Cells after MF and TKI Administration

We treated RCC4 and 786-O cells with increasing doses of MF, SUT, or AX. Figure 1 shows relative cell viabilities – IC50 values (defined as 50% relative cell viability) were determined by linear regression and extrapolation. For MF (Fig. 1a, b), MTS assays revealed IC50 values of 23.8 mm for RCC4 and 28.4 mm for 786-O cells. Regarding SUT (Fig. 1c, d), we measured IC50 values of 3.1 μm for RCC4 and 4.5 μm for 786-O cells. IC50 values for AX (Fig. 1e, f) were 2.9 μm (RCC4) and 2.4 μm (786-O), respectively.

Fig. 1.

MTS assays determining relative cell viabilities (in %) for RCC4 and 786-O cells treated with MF (a, b), SUT (c, d), and AX (e, f) for 120 h compared to untreated cells. IC50 values were calculated by using linear regression modeling.

Fig. 1.

MTS assays determining relative cell viabilities (in %) for RCC4 and 786-O cells treated with MF (a, b), SUT (c, d), and AX (e, f) for 120 h compared to untreated cells. IC50 values were calculated by using linear regression modeling.

Close modal

Combining MF and TKI Treatment Revealed Synergistic Effects

As illustrated in Figure 2a, single treatment with MF (10 mm) did not significantly alter the viability of RCC4 and 786-O cells, whereas SUT (2.5 μm) significantly diminished the viability of RCC4 cells. In contrast, identical doses of SUT did not significantly change cell viability of 786-O cells. Treating both cell lines with 2 μm AX (Fig. 2b) significantly diminished the viability of RCC4 and 786-O cells. Of note, adding the identical dose of MF (10 mm) to SUT or AX (Fig. 2a, b) augmented TKI effects in RCC4 and 786-O cells – resulting in a significantly lower proportion of viable cells compared to single TKI treatment.

Fig. 2.

Synergistic effects for MF and SUT/AX in RCC4 and 786-O cells. MTS assay results (120 h after treatment) for RCC4 and 786-O cells treated with MF, SUT (a) or AX (b) and a combination of MF and SUT/AX compared to EtOH-treated control cells (vehicle). **p < 0.01.

Fig. 2.

Synergistic effects for MF and SUT/AX in RCC4 and 786-O cells. MTS assay results (120 h after treatment) for RCC4 and 786-O cells treated with MF, SUT (a) or AX (b) and a combination of MF and SUT/AX compared to EtOH-treated control cells (vehicle). **p < 0.01.

Close modal

MF Regulated Expression of miR-205 and VEGFA in ccRCC Cells

As shown in Figure 3a and b, treating RCC4 and 786-O cells with MF (20 mm) caused a highly significant miR-205 overexpression along with a decreased expression of VEGFA. Western blotting confirmed decreased VEGFA protein levels after treatment with 20 mm MF for both RCC4 and 786-O cells (Fig. 3c). To examine the role of miR-205 and its regulation of VEGFA in ccRCC cells, we used a Tet-On model of miR-205 overexpression (Fig. 3d). For this, qRT-PCR experiments (Fig. 3e, f) affirmed significant overexpression of miR-205 after doxycycline treatment and a significant VEGFA downregulation. Western blotting revealed decreased VEGFA protein levels in the miR-205 Tet-On model (Fig. 3g). These data suggest that decreased VEGFA expression after MF treatment was at least partially caused by MF-driven miR-205 overexpression.

Fig. 3.

MF induces miR-205 expression with subsequent downregulation of VEGFA in RCC4 and 786-O cells – mimicking the effects on miR-205 and VEGFA expression in a doxycycline-inducible model of miR-205 overexpression (Tet-On model). a, b qRT-PCR confirmed MF-dependent miR-205 upregulation with subsequent VEGFA in both cell lines. c Western blotting yielded a progressive downregulation of VEGFA protein levels with increasing doses of MF (10 mm and 20 mm) in RCC4 and 786-O cells. d Tet-On model for miR-205 overexpression. e, f qRT-PCR experiments determining miR-205 overexpression and VEGFA downregulation within the miR-205 Tet-On model for RCC4 and 786-O cells. g Diminished VEGFA protein expression in miR-205 overexpressing RCC4 and 786-O cells (Tet-On model). c, g Vinculin was taken as housekeeping protein. **p < 0.01, ***p < 0.001.

Fig. 3.

MF induces miR-205 expression with subsequent downregulation of VEGFA in RCC4 and 786-O cells – mimicking the effects on miR-205 and VEGFA expression in a doxycycline-inducible model of miR-205 overexpression (Tet-On model). a, b qRT-PCR confirmed MF-dependent miR-205 upregulation with subsequent VEGFA in both cell lines. c Western blotting yielded a progressive downregulation of VEGFA protein levels with increasing doses of MF (10 mm and 20 mm) in RCC4 and 786-O cells. d Tet-On model for miR-205 overexpression. e, f qRT-PCR experiments determining miR-205 overexpression and VEGFA downregulation within the miR-205 Tet-On model for RCC4 and 786-O cells. g Diminished VEGFA protein expression in miR-205 overexpressing RCC4 and 786-O cells (Tet-On model). c, g Vinculin was taken as housekeeping protein. **p < 0.01, ***p < 0.001.

Close modal

Supernatant of miR-205-Overexpressing ccRCC Cells Reduced HUVEC Vessel Sprouting

Treating HUVECs with supernatants of miR-205 overexpressing ccRCC cells dramatically reduced their sprouting ability (Fig. 4a) – in terms of less endothelial sprouts (Fig. 4b) and shorter sprouts (Fig. 4c). These results indicate that high levels of miR-205 in ccRCC cells severely impede angiogenesis initiation in endothelial cells.

Fig. 4.

Number and average length of sprouts from HUVEC cells treated with the supernatant of miR-205-overexpressing ccRCC cells (Tet-On). a As illustrated by microscopic images (××40 magnification), doxycycline induction (i.e., miR-205 overexpression) compared to EtOH administration was associated with less (b) and shorter vessel sprouts – measured in pixel length by ImageJ (c). Presence and absence of recombinant VEGF within M200 medium served as positive and negative control. **p < 0.01. Method: cells treated with either EtOH or DOX were cultured overnight in hanging drops → incubated for 24 h → spheroids fixed and washed → pictures taken.

Fig. 4.

Number and average length of sprouts from HUVEC cells treated with the supernatant of miR-205-overexpressing ccRCC cells (Tet-On). a As illustrated by microscopic images (××40 magnification), doxycycline induction (i.e., miR-205 overexpression) compared to EtOH administration was associated with less (b) and shorter vessel sprouts – measured in pixel length by ImageJ (c). Presence and absence of recombinant VEGF within M200 medium served as positive and negative control. **p < 0.01. Method: cells treated with either EtOH or DOX were cultured overnight in hanging drops → incubated for 24 h → spheroids fixed and washed → pictures taken.

Close modal

miR-205 and VEGFA Expression in RCC Tissue

Next, we analyzed the expression of miR-205 and VEGFA in an in-house RCC cohort (n = 32) with corresponding malignant and adjacent benign tissue. qRT-PCR analyses revealed significantly lower expression levels of miR-205 (median ΔCt −3.94 in benign tissue vs. ΔCt −6.35 in tumors, p < 0.01, Fig. 5a) and significantly higher levels of VEGFA in cancer tissue (median ΔCt −1.47 in benign tissue vs. ΔCt −0.04 in tumors, p < 0.01, Fig. 5b). In line with the in-house cohort, the TCGA database (Fig. 5c) also revealed a downregulation of miR-205 accompanied by a VEGFA upregulation (Pearson’s ρ = −0.22, p < 0.01).

Fig. 5.

miR-205 and VEGFA expression in RCC. a miR-205 is significantly downregulated in malignant tissue of an in-house RCC cohort. bVEGFA is significantly upregulated in cancerous lesions. c Data from the TCGA ccRCC cohort: VEGFA is significantly overexpressed in malignant tissue (n = 523) compared to benign tissue (n = 100). Apart from VEGFA upregulation, miR-205 is significantly downregulated in ccRCC specimens – resulting in a significant negative correlation of VEGFA and miR-205 expression. FPKM, fragments per kilobase per million mapped reads; RPM, reads per million mapped reads.

Fig. 5.

miR-205 and VEGFA expression in RCC. a miR-205 is significantly downregulated in malignant tissue of an in-house RCC cohort. bVEGFA is significantly upregulated in cancerous lesions. c Data from the TCGA ccRCC cohort: VEGFA is significantly overexpressed in malignant tissue (n = 523) compared to benign tissue (n = 100). Apart from VEGFA upregulation, miR-205 is significantly downregulated in ccRCC specimens – resulting in a significant negative correlation of VEGFA and miR-205 expression. FPKM, fragments per kilobase per million mapped reads; RPM, reads per million mapped reads.

Close modal

VHL Influences miR-205 and VEGFA Expression as well as Drug Sensitivity of ccRCC Cells

To determine a potential influence of VHL on miR-205 and VEGFA expression, we used a constitutive model for VHLwt overexpressing clones of originally VHLmut RCC4 and 786-O cells. qRT-PCR analyses compared EV and VHLwt RCC4 and 786-O cells in terms of miR-205 (Fig. 6a) and VEGFA expression (Fig. 6b), thereby confirming a significant upregulation of miR-205 in VHLwt RCC4 and 786-O cells. Correspondingly, VEGFA was significantly lower in VHLwt-expressing ccRCC cell lines. Immortalized, nonmalignant HK-2 cells served as negative control and displayed significantly higher miR-205 as well as lower VEGFA expression compared to ccRCC cells. Western blotting (Fig. 6c) showed markedly decreased protein amounts of VEGFA in VHLwt-expressing RCC4 and 786-O cells. In the following, cell viability assays revealed a significantly higher sensitivity of VHLwt RCC4 and 786-O cells toward MF and SUT (Fig. 6d) compared to VHLmut parental RCC4 and 786-O cells.

Fig. 6.

Expression of miR-205 and VEGFA in RCC4 and 786-O cells depending on VHL status. a, bVHLwt RCC4 and 786-O cells exhibited higher levels of miR-205 and correspondingly lower levels of VEGFA. HK-2 cells were taken as control. cVHLwt RCC4 and 786-O cells were characterized by decreased VEGF protein levels. Vinculin served as a housekeeping protein. dVHLwt RCC cells were significantly more sensitive toward MF and SUT treatment. **p < 0.01.

Fig. 6.

Expression of miR-205 and VEGFA in RCC4 and 786-O cells depending on VHL status. a, bVHLwt RCC4 and 786-O cells exhibited higher levels of miR-205 and correspondingly lower levels of VEGFA. HK-2 cells were taken as control. cVHLwt RCC4 and 786-O cells were characterized by decreased VEGF protein levels. Vinculin served as a housekeeping protein. dVHLwt RCC cells were significantly more sensitive toward MF and SUT treatment. **p < 0.01.

Close modal

Growing evidence suggests tumor-suppressive effects of MF in various malignancies [1]. From a clinical perspective, regular MF intake was associated with prolonged overall survival in ccRCC patients treated with SUT [5, 6]. Functionally, findings from MF-treated murine embryonic fibroblasts [14] suggested that MF can upregulate cellular miR-205 expression. Therefore, we investigated whether MF also modulated miR-205 expression in ccRCC cells. Given the miR-205-mediated regulation of VEGFA in entities such as breast cancer, ovarian cancer, glioblastoma multiforme, and ccRCC itself [16, 23‒25], we examined whether MF-caused regulation of miR-205 expression was sufficient to modulate VEGFA expression in ccRCC cells.

MF Induced miR-205 Expression with Subsequent Targeting of VEGFA in ccRCC Cells

The IC50 values for MF were markedly higher for RCC4 and 786-O cells compared to IC50 counts reported previously for colon carcinoma [26] and prostate cancer cell lines [27]. Still, these values were substantially lower than those previously found for MF treatment in nonmalignant HK-2 cells [28]. Regarding SUT treatment of HK-2 cells, researchers also reported higher IC50 counts [29, 30] compared to SUT treatment of RCC4 and 786-O cells shown here (see online suppl. Table S2).

Regarding dual treatment of RCC4 and 786-O cells, we observed a significant synergistic effect for the combination of MF and TKI treatment with SUT or AX for both cell lines. Additionally, MF administration significantly induced the expression of miR-205 in both ccRCC cell lines. Apart from this specific trait, our Tet-On model confirmed that elevated miR-205 expression regulated the expression of VEGFA in RCC4 and 786-O cells. In addition, analyses of ccRCC specimens of an in-house RCC cohort and the TCGA database (KIRC cohort) revealed a downregulation of miR-205 in ccRCC tissue along with an upregulation of VEGFA.

In previous literature, MF was reported to act in both directions – either as antiangiogenic [31, 32] or as pro-angiogenic compound by, among other things, upregulating VEGFA [33, 34]. However, MF-mediated induction of miR-205 with subsequent decrease in VEGFA expression clearly represents a novel tumor suppressive and antiangiogenic trait of MF in ccRCC cells.

Impact of miR-205 Overexpressing ccRCC Cells on Angiogenesis Initiation in HUVEC Cells

We now wanted to take a closer look at the potential impact of MF on angiogenesis/vessel sprouting in vitro. For this reason, we performed HUVEC sprouting assays to determine an influence of the MF/miR-205/VEGFA axis. The analysis of HUVEC clusters treated with the medium supernatant of miR-205-overexpressing ccRCC cells revealed significantly less vessel sprouts. Moreover, sprouts from HUVEC cells treated with the supernatant from doxycycline-induced cells were significantly shorter. As miR-205 can regulate the expression of a variety of target genes, this experiment could not show an exclusive effect of the miR-205/VEGFA axis. However, targeting of VEGFA could at least partly explain the antiangiogenic effect of miR-205 observed. Taken together, the results at the very least strengthen the angiogenesis-inhibiting role of MF and miR-205 in ccRCC cells.

VHL Status Modulated the miR-205/VEGFA Axis in ccRCC Cells

In general, loss of VHL function activates hypoxia-inducible factor (HIF) signaling, leading to an increased expression of angiogenic growth factors [18, 19]. Given the crucial role of neo-angiogenesis in ccRCC, it is not astonishing that VHL gene defects appear in ∼75% of sporadic ccRCC [35]. For this reason, we were interested in whether the MF/miR-205/VEGFA axis was controlled by the VHL status of the ccRCC cells and compared the originally VHLmut RCC4 and 786-O cell lines with VHLwt RCC4 and 786-O cells. Of note, VHL status significantly determined miR-205 and VEGFA expression in RCC4 and 786-O cells. Our results suggest that miR-205 should be regarded as a VHL-regulated miR, a trait previously known for candidates such as miR-210 and miR-182-5p [36]. Moreover, the intersection of MF- and VHL-dependent angiogenic signaling strengthens the protective and antiangiogenic role of MF in ccRCC cells.

To strengthen the rationale for adding MF to antiangiogenic therapies in patients with ccRCC, additional experiments could be performed. Further analyzing the effect of MF on VHL-mediated, HIF-related signaling and its downstream targets could provide valuable insights regarding TKI interactions. Given that MF was already shown to regulate HIF1ɑ [37], this research could pave the way for a similar interaction of MF with HIF2ɑ inhibitors like belzutifan, which are currently tested in clinical trials [38, 39]. Of note, miR-205 expression itself appears to be regulated by HIF-mediated signaling [40]. This trait highlights the need for a better understanding of miR networks, as other miRs like miR-126, which also serves as a biomarker candidate in ccRCC [41], were shown to regulate HIF1a in turn [42]. Apart from counteracting angiogenic signaling, other MF effects such as inhibition of mitochondrial function could also contribute to the clinically observed added value of MF in TKI trials – as recent findings suggest that mitochondrial signaling could also play a crucial role in ccRCC [43].

Our results shed light on novel aspects of MF as a tumor suppressor in ccRCC cells. MF-mediated induction of miR-205 with subsequent downregulation of its target gene VEGFA could be one explanatory approach for the clinical synergism of MF and TKI in cancer patients. Of note, the MF/miR-205/VEGFA axis was critically influenced by the VHL status of the cancer cells. Clinically, our data provide a further rationale for examining MF in ccRCC trials.

The study was approved by the Ethical Review Board of the Julius Maximilians University Würzburg, Germany (No. 136/08), and written informed consent was obtained from all patients participating.

The authors have no conflicts of interest to declare.

M.K. was funded by a personal grant from Else-Kröner-Foundation (Else Kröner Integrative Clinician Scientist College for Translational Immunology, University Hospital Würzburg, Germany). Pfizer (New York, NY, USA) kindly provided sunitinib and axitinib.

Conceptualization: M.K., J.F., B.K., A.S., and C.K.; methodology: M.K., B.K., A.S., and C.K.; software and visualization: F.R., A.M., and C.K.; validation: H.K., B.K., and A.S.; formal analysis: M.K., A.M., S.J.R., and C.K.; investigation: M.J.K., J.F., F.R., S.J.R., A.M., and C.K.; resources: F.R., S.J.R., and C.K.; data curation: C.K.; writing: M.K., M.J.K., and C.K.; writing – review and editing: M.K., A.M., M.J.K., and C.K.; supervision: H.K., A.S., and C.K.; project administration: C.K.; and funding acquisition: H.K., B.K., A.S., and C.K.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author. The results shown here are in part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.

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