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

Abstract

Background: Germline mutations in the FLCN gene are responsible for the development of fibrofolliculomas, lung cysts and renal neoplasia in Birt-Hogg-Dube' (BHD) syndrome. The encoded protein folliculin (FLCN) is conserved across species but contains no classic motifs or domains and its function remains unknown. Somatic mutations or loss of heterozygosity in the remaining wild type copy of the FLCN gene have been found in renal tumors from BHD patients suggesting that FLCN is a classic tumor suppressor gene.

Results: To examine the tumor suppressor function of FLCN, wild-type or mutant FLCN (H255R) was stably expressed in a FLCN-null renal tumor cell line, UOK257, derived from a BHD patient. When these cells were injected into nude mice, tumor development was inversely dependent upon the level of wild-type FLCN expression. We identified genes that were differentially expressed in the cell lines with or without wild-type FLCN, many of which are involved in TGF-beta signaling, including TGF-beta2 (TGFB2), inhibin beta A chain (INHBA), thrombospondin 1 (THBS1), gremlin (GREM1), and SMAD3. In support of the in vitro data, TGFB2, INHBA, THBS1 and SMAD3 expression levels were significantly lower in BHD-associated renal tumors compared with normal kidney tissue. Although receptor mediated SMAD phosphorylation was not affected, basal and maximal TGF-beta-induced levels of TGFB2, INHBA and SMAD7 were dramatically reduced in FLCN-null cells compared with FLCN-restored cells. Secreted TGF-beta2 and activin A (homo-dimer of INHBA) protein levels were also lower in FLCN-null cells compared with FLCN-restored cells. Consistent with a growth suppressive function, activin A (but not TGF-beta2) completely suppressed anchorage-independent growth of FLCN-null UOK257 cells.

Conclusions: Our data demonstrate a role for FLCN in the regulation of key molecules in TGF-beta signaling and confirm deregulation of their expression in BHD-associated renal tumors. Thus, deregulation of genes involved in TGF-beta signaling by FLCN inactivation is likely to be an important step for tumorigenesis in BHD syndrome.

Figure 1

Characterization of the UOK257 cell lines. (A) FLCN protein expression in the UOK257 cell lines restored with wildtype or mutant FLCN. P, parental; HR, FLCN (H255R) mutant; 2, 3, 4 and 6, wildtype FLCN. (B) FLCN mRNA level was measured by quantitative RT-PCR. Columns, mean; bars, +SD (n = 3). (C) Cell growth of the UOK257 cell lines in vitro. Points, mean (n = 4). (D) Colony formation assay of the UOK257 cell lines and control HT1080 cell line in soft-agar culture. Columns, mean; bars, +SD (n = 3).

Figure 2

Suppression of tumorigenesis by re-introduction of wild-type FLCN not mutant FLCN in the FLCN-null cell line, UOK257. (A) Tumorigenesis of UOK257 cells in nude mice was suppressed by wild-type FLCN. Arrow indicates a tumor from UOK257-P cells. Arrowhead indicates a flat patch of tumor cells from UOK257-3 cells. (B) Histologies of the tumors from different clones of UOK257 cells. (a) high, (b) medium, and (c) low grade clear cell histologies; (d) papillary RCC.

Figure 3

Genes differentially expressed in mutant and wild-type FLCN cell lines. (A) Heat-map representation of the genes differentially expressed in mutant and wild-type FLCN cell lines, and hierarchical clustering of the cell lines. Gene symbols are listed on the left side of each row. Three prominent groups of genes involved in signaling pathways were indicated on the left side of the gene symbols. (B) Deregulation of the key molecules in TGF-β signaling by FLCN knockdown in the UOK257-2 cells expressing wildtype FLCN. C, control cell line infected with empty retrovirus; KD, FLCN-knockdown cell line infected with a retrovirus expressing short hairpin RNA targeting FLCN.

Figure 4

Genes involved in TGF-β signaling and the encoded proteins dysregulated in renal tumors from BHD patients. (A) Quantitative RT-PCR of TGFB2, INHBA, SMAD3, THBS1, GREM1 and FLCN expression levels in human BHD tumors (T, n = 12) and normal kidneys (N, n = 8). Gene expressions were normalized against cyclophilin gene expression and relative expressions were calculated against gene expression in UOK257 cells. Median values of expression levels were indicated with short lines. Points, mean expression of each sample. P value, Mann-Whittney U-test. (B) Reduced expression of TGF-β2 in the BHD tumors (left panel) and the UOK257 xenograft tumors (right panel) compared to normal renal tubules as shown by immunohistochemical staining. Five BHD tumors were examined and a representative immunostained image is shown. (C) Positive correlation of pSMAD3 and SMAD3 with FLCN expression in human BHD kidney tumors and normal kidney tissue. pSMAD3, SMAD2, SMAD3 and FLCN protein levels were measured by western blot analysis in normal kidney tissue (n = 5) and BHD tumors (n = 11).

Figure 5

TGF-β1 induced SMAD3 phosphorylation and effects of AICAR, Compound C and rapamycin on TGF- β1 induction of TGFB2 and INHBA in FLCN-null and FLCN-expressing cells. (A) TGF-β1 induced SMAD3 phosphorylation in UOK257 and UOK257-2 cells. Cells were treated with 0, 0.1 and 1 ng/ml of TGF-β1 for 1 hr. (B) TGF-β1 induced TGFB2, INHBA and SMAD7 mRNA expression in UOK257 and UOK257-2 cells. Cell were treated with 1 ng/ml of TGF-β1 for 0, 6, 12 and 24 hr after serum starvation for 12 hr. (C) (a and b) Cells were serum-starved for 24 hr and treated with 0.5 mM AICAR (AI), 20 μM Compound C (CC) or 1 nM rapamycin (Rapa) for 12 hr. UOK257-2/vector, UOK257-2 cells infected with empty retrovirus; UOK257-2/FLCN-KD, UOK257-2 cells infected with retrovirus expressing shRNA targeting FLCN. (c and d) Cells were serum-starved for 15 hr and treated with 0.5 mM AICAR or 20 μM Compound C (CC) for 6 hr.

Figure 6

Higher levels of secreted TGF-β2 and activin A proteins were detected in FLCN-restored UOK257-2 cells compared to the parental UOK257 cell line. (A) TGF-β2 and (B) activin A levels in cultured media were measured by ELISA (R&D systems). (C) TGF-β2 induced anchorage independent growth of UOK257 cells. However, (D) activin A suppressed anchorage independent growth of UOK257 cells. UOK257 cells (2,500 cells) were plated in soft-agar and cultured for 4 weeks in the presence of TGF-β2 or Activin A and stained with crystal violet. N = 4 for each treatment.