Characterizing the tumor suppressor activity of FLCN in Birt-Hogg-Dubé syndrome cell models through transcriptomic and proteomic analysis

Abstract

Birt-Hogg-Dubé syndrome (BHD) patients are uniquely susceptible to all renal tumor subtypes. However, the underlying mechanism of carcinogenesis is unclear. To study cancer development in BHD, we used human proximal kidney (HK2) cells and found that long-term folliculin (FLCN) knockdown was required to increase the tumorigenic potential of these cells, as evidenced by the formation of larger spheroids under nonadherent conditions. Transcriptomic and proteomic analyses revealed links between the FLCN, cell cycle control and DNA damage response (DDR) machinery. In addition, HK2 cells lacking FLCN had an altered transcriptome profile and enriched cell cycle control genes. G₁/S cell cycle checkpoint signaling was compromised by increased protein levels of cyclin D1 (CCND1) and hyperphosphorylation of retinoblastoma 1 (RB1). A FLCN interactome screen revealed that FLCN binds to DNA-dependent protein kinase (DNA-PK). This novel interaction was reversed in an irradiation-responsive manner. Knockdown of FLCN in HK2 cells caused a marked increase in γH2AX and RB1 phosphorylation. The levels of both CCND1 and phosphorylated RB1 remained high during DNA damage, which was associated with defective cell cycle control caused by FLCN knockdown. Furthermore, Flcn-knockdown C. elegans were defective in cell cycle arrest caused by DNA damage. This work revealed that long-term FLCN loss and associated cell cycle defects in BHD patients could contribute to their increased risk of cancer.

Fig. 1. Long-term knockdown of FLCN increases tumorigenesis.

A HK2 cells with or without short-term or long-term FLCN shRNA knockdown (compared to cells with nontargeting shRNA) were grown in soft agar for 21 days. The colony diameter was measured using ImageJ. (v1.53t), and a representative image of the tumors is shown (scalebar 250 μm), with the distribution of the tumors graphed. n = 360 per condition over 6 biological samples. Each data point represents a single tumor. Stats: data were not normally distributed (according to D’Agostino and Pearson tests) and were analyzed using nonparametric Kruskai-Wallis ANOVA with Dunn’s multiple comparisons test. B These HK2 cells were also plated under nonadherent conditions and imaged over 14 days (scalebar 250 μm), and the percentage change in diameter was plotted over time (n = 42). C RNA sequencing of HK2 cells with short- or long-term nontargeting or FLCN shRNA knockdown was carried out (n = 3). Differential gene expression was compared between short-term FLCN knockdown and nontarget shRNA knockdown and long-term FLCN knockdown and nontarget shRNA knockdown HK2 cells. Volcano plots are shown with the following thresholds: ≤ & ≥ 2.5-fold change and padj < 0.05 with false rate discovery (FDR) correction applied.

Fig. 2. Differentially expressed genes linked to E2F and known FLCN regulatory genes after FLCN knockdown.

Differential gene expression of E2F genes was compared between A short-term FLCN knockdown cells and nontarget shRNA knockdown cells and B long-term FLCN knockdown and nontarget shRNA knockdown HK2 cells after RNA sequencing. Volcano plots are shown with the following thresholds: ≤ & ≥ 2.5-fold change and padj < 0.05 with false rate discovery (FDR) correction applied. C FLCN-linked genes from this RNA sequencing experiment are graphed and include FLCN, CCND1, TP53, PPARGC1A, TGFA, CDKN1A, SQSTM1 and CCNE1.

Fig. 3. Differentially expressed genes and their associated signaling pathways.

A RNA sequencing was used to compare long-term FLCN shRNA-mediated knockdown and nontargeting shRNA-mediated knockdown in HK2 cells (scale is set to log2-fold change). Each gene is depicted in a signaling flow diagram. The known signaling functions of FLCN include (i) enhancing HIF-1α activity, as shown by increased expression of the HIF-1α target genes SLC2A2 and REDD1. A signaling feedback mechanism reduces HIF-1α through reducing HIF-1α expression and upregulating SKP2, a HIF-1α inhibitor. (ii) Upregulation of TGFβ/SMAD3 signaling upon knockdown of FLCN. SMAD3 is an inhibitor of the CCND1-CDK4/CDK6 complex. (iii) The expression of PGC1α and its downstream genes NR1H3, FOXO4, SOX9, CYCS, and HMOX1 was markedly upregulated upon FLCN knockdown. (iv) The INK4 family members that inhibit CCND1 and CDKN2A-C are increased, while CDKN2D is reduced. (v) Reduced expression of CDKN1A inhibits both CCND1 and CCNE1 activity. This finding demonstrated the presence of a transcriptional feedback mechanism that enhances CDK4/6 and CDK2 activity. Further differences were observed with enhanced CDK6 expression and decreased expression of CCND1 and CCNE1. B Relative gene expression of cyclin-dependent kinase inhibitors across the different HK2 cell lines was calculated (n = 3). C Protein lysates from spheroids generated from HK2 cells with or without short- or long-term FLCN shRNA knockdown (nontargeted shRNA was used as a control) were probed for phosphorylated RB1, CCND1, TP53, and CDKN1A; phosphorylated AMPK; and ACC; SQSTM1, β-catenin, FLCN, and β-actin, which were used as loading controls (n = 3).

Fig. 4. FLCN has an extensive PPI network.

A GST-FLCN was overexpressed in HEK293 cells, after which the purified and interacting proteins were separated via SDS‒PAGE and stained with colloidal blue. The gel was sectioned for mass spectrometry analysis. B The FLCN protein interaction network is shown, which includes known FLCN interactions. The total peptide sequences identified after mass spectrometry are indicated. C FLCN-binding proteins identified by mass spectrometry were analyzed by DAVID, and the top ten enriched scored biological processes or cellular components are presented. D FLCN-binding proteins are involved in protein folding and complex formation, metabolism and mTOR signaling, and cell cycle and DNA damage.

Fig. 5. FLCN interacts with DNA-PK.

A GST-tagged FLCN was overexpressed in HEK293 cells and used as a bait protein to validate the interactions between FLCN and endogenously expressed DNA damage components (DNA-PKcs, ATM, and ATR) via GST pull-down. B Endogenous FLCN was immunoprecipitated using an antibody against N-terminal FLCN, and the interaction of endogenous DNA-PKcs was detected via western blotting. C HA-tagged FLCN constructs (wild-type FLCN (WT)) and two patient-derived C-terminal truncation mutants (Y463X and H429X) were overexpressed in HEK293 cells and immunoprecipitated using anti-HA antibodies, and bound endogenous DNA-PKcs was detected via western blotting. D DNA-PK kinase assays were performed using GST-FLCN or GST-TP53, which were overexpressed and purified from HEK293 cells. The incorporation of radiolabeled phosphate [³²P] was determined with active DNA-PK, which was further induced by supplementation with short double-stranded DNA (dsDNA).

Fig. 6. FLCN knockdown promotes cell cycle progression.

A Serine 139 phosphorylation of the histone variant H2AX (γH2AX) was assessed under basal conditions in HK2 cells grown in standard cell culture with and without short-term or long-term FLCN shRNA knockdown (nontargeted shRNA was used as a control). B The FLCN/DNA-PKcs interaction was investigated following IR-induced DNA damage. GST-tagged FLCN was overexpressed in HEK293 cells that were then subjected to IR (5 or 10 Gy) and left for either 1 or 4 h prior to cellular lysis, as indicated. GST-FLCN was purified, and endogenous DNA-PKcs was detected via western blotting. C In HK2 cells with or without short- or long-term FLCN shRNA knockdown, DNA damage markers were assessed following 5 Gy IR for 1 h, where indicated. γH2AX, P-DNA-PKcs (Ser2056) and the downstream DNA-PK substrate P-TP53 (Ser15) are shown. G₁/S phase cell cycle markers CCND1 and P-RB1 were analyzed and FLCN and β-actin were used as controls. D Mitotic germ cells were quantified in C. elegans with and without Flcn siRNA knockdown and subjected to UV damage, where indicated. Mitotic germ cells were scored and graphed. Representative images of the mitotic cells are presented (n = 18).