Tumor suppressor FLCN inhibits tumorigenesis of a FLCN-null renal cancer cell line and regulates expression of key molecules in TGF-β signaling

To evaluate the tumor suppressor function of FLCN, wild-type or mutant (H255R) FLCN cDNA was introduced into the FLCN-null UOK257 cells using lentiviral vectors. Four clones expressing wild-type FLCN (UOK257-2, -3, -4, and -6) and one clone expressing mutant FLCN-H255R (UOK257-H255R; Flcn missense mutation responsible for canine RCND) were isolated and compared to the parental UOK257 cells (UOK257-P). FLCN protein expression was measured by Western blot analysis using a mouse monoclonal anti-FLCN antibody. Relatively high levels of FLCN protein were detected in the UOK257-2, -4 and -6 cells but very low levels of FLCN protein were detected in the UOK257-3 and UOK257-H255R cells (Fig. 1A). FLCN mRNA expression from both the transgene and endogenous FLCN was measured by quantitative RT-PCR. The total FLCN mRNA expression was increased by the expression of the wild-type FLCN or mutant FLCN-H255R transgene to varying degrees in the cell lines (P = 3 < 4 < H255R <2 <6) (Fig. 1B).

We examined whether introduction of wild-type or mutant FLCN could affect anchorage dependent and independent growth of UOK257 cells. Anchorage dependent growth of UOK257 cells on culture dishes was not affected by the expression of wild-type or mutant FLCN (Fig. 1C). However, anchorage independent growth measured as colony numbers on soft agar was low in the wild-type FLCN cell line UOK257-2, which expressed high levels of FLCN, compared to the parental UOK257 cell line (UOK257-P) (Fig. 1D). One of the characteristics of the UOK257 cells was a slow growth rate (doubling time, 52+/- 9 hrs) on culture dishes. These cells also grew very slowly in soft agar taking 3-4 weeks to reach a countable colony size. By comparison, HT-1080 cells derived from a fibrosarcoma grew faster in soft agar and often generated larger colonies (data not shown).

To examine whether the tumorigenic potential of UOK257 cells was affected by wild-type or mutant FLCN, mutant FLCN (UOK257-P and -H255R) and wild-type FLCN-expressing cells (UOK257-2, -3, -4 and -6) were injected subcutaneously with matrigel into athymic nude mice and tumor growth was measured for up to one year. Most of the mice injected with UOK257-P and UOK257-H255R cells developed solid tumors, although some animals only developed flat patches of tumor cells (See additional file 1: Table S1 and Fig. 2A). All of these tumors were high grade and exhibited clear cell histology (See additional file 1: Table S1 and Fig. 2B). On the other hand, the mice injected with UOK257 cells expressing a high level of FLCN (UOK257-2, -4 and -6) did not develop tumors (See additional file 1: Table S1 and Fig. 2A). Instead, flat masses of cells only rarely containing tumor cells were observed in 6 of 35 (17%) of the animals. The mice injected with UOK257-3 cells expressing a very low level of FLCN developed solid tumors with low incidence (2/10) and smaller size (See additional file 1: Table S1). In some animals, UOK257-3 cells grew as flat patches (5/10; Fig. 2A) and exhibited mostly clear cell histology with varying grades (low to high; Fig. 2B).

We investigated whether wild-type or mutant FLCN transgenes, or the endogenous mutant FLCN genes were lost during tumor progression. Genomic DNA was isolated from the tumors or tumor cell patches and PCR was performed using a primer pair specific to exon 10 and exon 11 that amplifies 664 bp of the endogenous FLCN gene or 99 bp of the FLCN transgene. All of the tumors from the cell lines retained the endogenous mutant FLCN gene (c.1285dupC) and all of the tumors from UOK257-3 and UOK257-H255R retained their respective transgenes (See additional file 1: Fig. S1).

To identify the genes regulated by FLCN expression, we performed gene expression microarray analysis using RNAs isolated from the UOK257 cell lines expressing either no, mutant or wild-type FLCN. We identified a total of 439 genes, which were up or down-regulated more than 2-fold in the mutant and FLCN-null cell lines (UOK257-P and UOK257-H255R) compared to the wild-type FLCN cell lines (UOK257-2, -3, -4 and -6) (See additional file 2: Table S2). To explore the biological processes and pathways regulated by FLCN, the genes were subclassified with the help of "Panther Classification System" http://​www.​pantherdb.​org and three prominent pathways were identified, namely cadherin signaling, TGF-β signaling, and angiogenesis (Fig. 3A). Although all three of these pathways are important in tumorigenesis, we focused on the genes involved in TGF-β signaling (See additional file 1: Fig. S2). We found that TGF-β2 (TGFB2), Inhibin β A (INHBA, a subunit of activin A), SMAD3 (SMAD3) and thrombospondin-1 (THBS1) were down-regulated, and Gremlin (GREM1) was upregulated in FLCN-null and mutant FLCN-H255R UOK257 cells compared with FLCN-restored UOK257 cells. We confirmed the GREM1, TGFB2, INHBA, SMAD3 and THBS1 microarray results by quantitative RT-PCR (See additional file 1: Fig. S3).

We next examined whether the expression levels of TGFB2, INHBA, THBS1, GREM1 and SMAD3 could be deregulated by knockdown of FLCN in FLCN-restored UOK257 cells. A FLCN- knockdown cell line was generated by introducing a retrovirus that expressed shRNA against FLCN in FLCN-restored cells (UOK257-2). In addition to reduced expression of FLCN, the expression of TGFB2, INHBA, THBS1 and SMAD3 was decreased and the expression of GREM1 was increased in the FLCN-knockdown cell line (Fig. 3B).

In order to determine whether the genes that were regulated by FLCN in in vitro cell culture were differentially expressed in renal tumors from BHD patients compared to normal kidney parenchyma, we performed quantitative RT-PCR using RNA isolated from these tissues. GREM1, TGFB2, INHBA, THBS1 and SMAD3 RNA expression levels were significantly lower in the BHD renal tumors compared to normal kidney tissue (Fig. 4A). However, FLCN RNA levels were not statistically different (P = 0.316). In support of the RT-PCR data, immunohistochemical staining of TGF-β2 showed strong TGF-β2 expression in the normal renal tubules but reduced expression in the tumors from BHD patients (Fig. 4B, left panel). In addition, the UOK257 xenograft tumors expressed lower levels of TGFB2 compared to normal mouse kidney (Fig. 4B, right panel).

We measured protein expression of SMAD2, SMAD3, phospho-SMAD3 (pSMAD3) and FLCN in renal tumors from BHD patients (n = 11) and normal human kidney tissue (n = 5). pSMAD3 levels were high in 3 out of 5 normal kidneys but only 1 (T11) of 11 tumors (Fig. 4C). In addition, SMAD3 levels and SMAD3/SMAD2 ratios were higher in normal kidneys compared to the tumors. On the other hand FLCN protein levels were lower or undetectable in all tumors except T11, in which a moderate level of FLCN expression was detected along with high levels of pSMAD3 and SMAD3 (Fig. 4C). Therefore it is likely that the T11 tumor was contaminated with normal kidney tissue.

In order to investigate whether receptor mediated TGF-β signaling was disrupted by the loss of FLCN expression, TGF-β induced SMAD3 phosphorylation was examined in UOK257 cells and compared to UOK257-2 cells. TGF-β induced SMAD3 phosphorylation was not affected by FLCN inactivation (Fig. 5A). In addition, BMP4 induced SMAD1/5/8 phosphorylation was not disrupted by loss of FLCN expression (data not shown). We then examined whether TGF-β induced gene expression was dysregulated in FLCN-null UOK257 cells. SMAD7, an inhibitory SMAD, is known to be induced by TGF-β. SMAD7 expression was induced in both UOK257 and UOK257-2 cell lines (Fig. 5B). However the basal and the maximal induced levels of SMAD7 were two fold greater in UOK257-2 cells than in UOK257 cells. Similar to SMAD7, TGFB2 and INHBA expressions were induced by TGF-β in both cell lines but their basal and maximal levels of expression were substantially higher (5.8- to 23-fold) in FLCN-restored UOK257-2 cells compared with FLCN-null UOK257 cells (Fig. 5B).

Since FLCN and FNIP1/2 can complex with AMPK, and phosphorylation of these proteins is affected by AMPK and mTOR signaling, we wanted to know whether AMPK and mTOR signaling affected the expression of TGFB2 and INHBA in a FLCN -dependent manner. Interestingly, both TGFB2 and INHBA were induced by the AMPK activator AICAR but reduced by the AMPK inhibitor Compound C in UOK257-2 cells expressing FLCN as well as in cells in which FLCN expression was knocked down by a retrovirus expressing FLCN shRNA [UOK257-2/FLCN-KD; Fig. 5C(a) and Fig. 5C(b)]. Rapamycin, an inhibitor of mTOR signaling, also induced TGFB2 and INHBA expression in both FLCN-expressing UOK257-2 and UOK257-2/FLCN-KD cells [Fig. 5C(a) and Fig. 5C(b)]. However, the basal and maximal levels of induction of TGFB2 and INHBA by AICAR and rapamycin were higher in FLCN-expressing UOK257-2 cells compared to UOK257-2/FLCN-KD cells [Fig. 5C(a) and Fig. 5C(b)]. Similar results were obtained from an experiment using UOK257 FLCN-null and UOK257-2 FLCN-expressing cell lines. Interestingly, AICAR induced TGFB2 and INHBA expression in UOK257-2 cells but not in UOK257 cells [Fig. 5C(c) and Fig. 5C(d)].

Initially, in order to confirm that protein expression levels were consistent with the mRNA expression levels, we measured secreted TGF-β2 and activin A levels in the media of UOK257 and UOK257-2 cells by ELISA. In accordance with their mRNA expression, TGF-β2 and activin A protein expression levels were lower in UOK257 cells compared to UOK257-2 cells (Fig. 6A and 6B). Since both TGF-β2 and activin A have been reported to suppress cell growth, we examined their effect on growth of UOK257 cells. To evaluate the growth suppressive effects of TGF-β2 and activin A, UOK257 cells were treated with TGF-β2 or activin A and cultured for 4 weeks in soft-agar. Unexpectedly, TGF-β2 appeared to increase colony formation of UOK257 cells at both 1 ng/ml and 5 ng/ml (Fig. 6C). However, activin A reduced colony formation at 1 ng/ml and completely suppressed colony formation at 5 ng/ml (Fig. 6D).

Wild-type or mutant (H255R) FLCN cDNA was transduced into UOK257 cells using the ViraPower Lentiviral expression system (Invitrogen) following the manufacturer's protocols. Stable clones were selected using Blasticidin S (1.5 μg/ml). Cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. To evaluate growth rate in culture, cells (2 × 10³) were plated in each well of five 96 well plates, cultured, and cell numbers were measured at day 1, 2, 3, 5 and 7 using the CyQuant Cell Proliferation Assay Kit (Molecular Probes). Adenoviral vectors (pAd/CMV/V5-DEST) expressing wild-type and mutant (c.1285dupC) FLCN were generated using the ViraPower Adenoviral Gateway system (Invitrogen) following the manufacturer's protocol. A retroviral shRNA vector targeting FLCN was generated by inserting double stranded oligonucleotides (forward sequence, 5'-GATCCCCGGTGTTTGAGGCAGAGCAGTTCAAGAGACTGCTCTGCCTCAAACACCTTTTTA-3' and reverse sequence, 5'-GCTTAAAAAGGTGTTTGAGGCAGAGCAGTCTCTTGAACTGCTCTGCCTCAAACACCGGG-3') into HindIII and BglII sites of pSuper-Retro vector (Oligoengine) following the manufacturer's instruction. UOK257-2 cells were infected with the FLCN shRNA vectors and selected against puromycin (7.5 ug/ml).

UOK257 cells (5 × 10³) were suspended in 1.5 ml of 0.3% agar in DMEM containing 10% FBS and were overlayed on 1.5 ml of pre-solidified 0.5% agar in the same medium. Cells were cultured in a CO₂ incubator for 3-4 weeks. Colonies were stained for 1 hour with 0.02% crystal violet solution dissolved in 10% neutral formalin. Colony number was counted under a dissection microscope after washing with PBS three times.

Cells (1 × 10⁶) suspended with basement membrane matrix (BD Biosciences) were injected subcutaneously into the flanks of athymic nude mice. Tumor growth was measured once a week and mouse health was monitored daily. Mice bearing tumors larger than 2 cm, or showing severe health problems, were sacrificed and examined. Otherwise tumor growth was monitored for up to one year after injection. Tumors were fixed in 10% buffered formalin solution for histological examination and flash frozen in liquid nitrogen for protein and RNA extraction. Animal care procedures followed NCI-Frederick Animal Care and Use Committee guidelines.

Cells were harvested and lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0 with 150 mM NaCl, 1.0% NP-40 and 0.5% sodium deoxycholate) or 1× SDS sample buffer (Biorad). Cell lysates were resolved by 4-20% SDS PAGE and blotted onto PVDF membrane. The following antibodies were used in this study: anti-FLCN mouse monoclonal [20], anti-β-actin (Sigma), anti-SMAD2/3 (Santa Cruz, sc-6032), and anti-pSMAD2/3 (Santa Cruz, sc-11769) antibodies. Immunoblots were processed by the ECL Detection System (Pierce) according to the manufacturer's protocols.

Paraffin tissue sections were deparaffinized, rehydrated in graded alcohol and boiled in Tris-EDTA buffer pH 8.0 for 20 min at 90°C for antigen retrieval. After blocking, sections were probed with primary antibodies overnight and then incubated with HRP-polymer conjugated secondary antibodies. Diaminobenzidine hydrochloride (DAB) was used as a substrate for peroxidase. Sections were then briefly counterstained with hematoxylin and permanently mounted for observation.

Cells (2 × 10⁵) were cultured on 6 well plates for 3 days and culture media was collected for assay. TGF-β2, and activin A levels in the media were quantified by Human TGF-β2 DuoSet (R&D systems) and activin A DuoSet (R&D systems), respectively, following the manufacturer's instruction.

Total RNAs were isolated from the UOK257 cell lines using Trizol reagent (Invitrogen) and further purified using RNeasy mini kit (QIAGEN) according to the manufacturer's protocols. Probes, which were generated using these RNAs, were hybridized to the Human Genome U133 Plus 2.0 arrays (Affymetrix) and processed according to recommended protocols. The CEL files were processed using the Partek Genomic Suite 6.2 (Partek Inc.). Data were transformed using a log normalization process and the differentially expressed genes were identified by Student's t-test and Mann-Whitney U-test. The genes that were differentially expressed in mutant FLCN cell lines (UOK257-P and -H255R) and wild-type FLCN cell lines (UOK257-2, -4 and -6) were used for further analysis.

To confirm the microarray results, quantitative real-time reverse transcription PCR (RT-PCR) was performed. RNAs were digested with DNase I for 30 min at 37°C followed by heat denaturation at 70°C for 20 min to remove genomic DNA contamination. Total RNAs (2.5 μg) were primed with 100 ng random primers and reverse-transcribed by Superscript II reverse transcriptase (Invitrogen) at 42°C for 1 hr. The identical reactions were performed without reverse transcriptase to generate negative controls. PCR primers were generated using Primer 3 software [41] or Primer Express 3.0 (Applied Biosystems). Quantitative RT-PCR was performed with Power SYBR-Green or Taqman Gene Expression Master Mix (Applied Biosystems) using a 7300 Real-Time PCR system (Applied Biosystems) following the manufacturer's protocols. All reactions were run in triplicate using β-actin, GAPDH or cyclophilin A genes as internal controls. The relative level of a particular gene expression was evaluated according to the function of 2^-ddCt, where ddCt is dCt_(treatment) - dCt_(control), dCt is Ct_{(target gene)} - Ct_{(GAPDH or actin)} and Ct is the cycle at which the threshold is crossed. The gene-specific primer pairs for the PCR reactions are as follows: FLCN forward 5'-TTCACGCCATTCCTACACCAGA-3' and reverse 5'-GCCCACAGGTTGTCATCACTTG-3', GREM1 forward 5'-GCAAAACCCAGCCGCTTAA-3' and reverse 5'-TGATGGTGCGACTGTTGCA-3', TGFB2 forward 5'-CGAGAGGAGCGACGAAGAGT-3' and reverse 5'-AGGGCGGCATGTCTATTTTG-3', THBS1 forward 5'-CCAGATCAGGCAGACACAGA-3' and reverse 5'-AGTTGTCCCGTTCATTGAGG-3', INHBA forward 5'-TGGAGTGTGATGGCAAGGTCA-3' and reverse 5'-GCATGATAGCCAGAGGGAGCA-3', SMAD3 forward 5'-GACGAGGTCTGCGTGAATCC-3' and reverse 5'-GTGGCGTGGCACCAACA-3', and GAPDH forward 5'-TTCCACCCATGGCAAATTCC-3' and reverse 5'-CGCCCCACTTGATTTTGGAG-3'. SMAD7 forward 5'-CCAACTGCAGACTGTCCAGA-3' and reverse 5'-CAGGCTCCAGAAGAAGTTGG-3'. PCR product quality was monitored using post-PCR dissociation curve analysis.